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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8033242. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: A human-specific motif facilitates CARD8 inflammasome activation after HIV-1 infection Jessie Kulsuptrakul, Elizabeth A. Turcotte, Michael Emerman* and Patrick S. Mitchell* doi: https://doi.org/10.1101/2022.10.04.510817 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029 SUMMARY: CARD8 is thought to sense infection stress and stimulate inflammasome formation, but precise mechanisms remain to be elucidated. HIV-1 protease (HIV-1PR) cleaves CARD8, resulting in proteasome-dependent degradation of the amino-terminal CARD8 fragment and liberation of the carboxy-terminal fragment that activates inflammasomes. Kulsuptrakul and colleagues build upon this intriguing finding to investigate evolutionary relationships between primate lentiviruses and CARD8 homologs from their host primates. They discovered that both HIV-1 and SIVcpzproteases cleave human CARD8. By contrast, chimpanzee CARD8 was not cleaved by primate lentivirus proteases. Thus, even prior to the HIV-1 pandemic, SIVcpz proteases could cleave and activate human CARD8. The authors provided evidence for CARD8-dependent inflammasome activation in an in vitro HIV-1 infection model. Complementation of CARD8 KO cells with WT human CARD8 restored inflammasome activation, whereas complementation with chimpanzee CARD8 or human CARD8 mutant constructs with substitutions that destroy the protease cleavage site did not. Together, these findings support a role for human CARD8 in activating the inflammasome upon HIV infection and suggest that, during spillover events, SIVcpz was well equipped to cleave human CARD8 and drive pathogenesis. OVERALL ASSESSMENT: This study convincingly demonstrates that human CARD8 is susceptible to cleavage and activation by diverse primate lentivirus protease enzymes, whereas chimpanzee CARD8 is not. Overall, the data is quite convincing and clearly organized and rendered for readers, and positions lentivirus proteases and host CARD8 sensors as new players in the 'genomes-in-conflict' chess match. These conclusions could be strengthened by additional evidence for CARD8-mediated inflammasome activation. STRENGTHS: The manuscript is well-written. The rationale provided for the investigation of CARD8 in the context of HIV infection is sound. The data presented is well-organized, clear and well-controlled. The primary strength is the identification and careful mapping of viral protease-mediated cleavage and activation of human CARD8, whereas the closely related chimpanzee CARD8 resists cleavage and activation. WEAKNESSES: While the manuscript was well written, the introduction was quite brief. We suggest that the authors should provide some additional information about CARD8 and inflammasomes. Does CARD8 have additional roles in the cell beyond inflammasome activation? How does CARD8 fit into the context of a diverse array of inflammasome triggers? This kind of information would really help set the stage properly. The data in Figs. 3/4 could be strengthened by additional assays to measure inflammasome activation beyond IL-1β secretion and PI staining. Options include an IL-18 ELISA, LDH release assay, HMGB1 release assay, or caspase-1 cleavage assay (western blot). The provision of a second assay to corroborate inflammasome activation is consistent with field-specific standards. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.5 · Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls. · Fig. 1B – the cartoon introduces a 2nd PR cleavage site on CARD8 (site 88/89) that is not investigated further. Is this site relevant to this study? Do the amino acids at this site vary between humans and Old-world monkeys? The reader would benefit from some explanation about this protease cleavage site. · Fig. 1C – We wondered whether treatment with a proteasome inhibitor could prevent degradation of CARD8 protein fragments and help us better understand cleavage patterns on western blots. Moreover, could the unknown band at ~40 kDa be further characterized, and does it relate to the N-terminal cleavage product? · Figs 1D/2B - The anti-vinculin loading control western blots for Figure 1D and Figure 2B are identical. · Fig. 3 – As mentioned above, abstract-level conclusions about inflammasome activation require better support. The data in Fig. 3 could be strengthened by additional assays to measure inflammasome activation beyond IL-1β secretion and PI staining. Options include an IL-18 ELISA, LDH release assay, HMGB1 release assay, or caspase-1 cleavage assay (western blot). The provision of a second assay to corroborate inflammasome activation is consistent with field-specific standards. · Figs. 4B/4C – "Similar to our observations with VbP, we found that IL-1β secretion and cell death were significantly reduced in Pam3CSK4-primed, HIV-1LAI or HIV-1LAI-VSVG infected CARD8 KO versus WT THP-1 cells (Figure 4C). These results indicate that HIV-1-induced inflammasome activation in THP-1 cells is dependent on CARD8." - This statement requires greater support demonstrating CARD8-dependent inflammasome activation. Inflammasome activation was still evident in the CARD8 KO cells. This analysis could benefit from corroboration with a second assay, as mentioned above. We wondered whether it would be feasible to employ a reconstructed inflammasome model, as reported in this recent paper: o Qiyao Chai et al. A bacterial phospholipid phosphatase inhibits host pyroptosis by hijacking ubiquitin. Science 378, eabq0132(2022). DOI:10.1126/science.abq0132 · Fig. 4D - We were once again curious about the identity of the unknown ~40 kDa band, which is clearly present in the CARD8 KO lysates. Perhaps this could be addressed in the Discussion. Are specific analyses performed using methods that are consistent with answering the specific question? · Is there appropriate technical expertise in the collection and analysis of data presented? · Yes · Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? · Yes · Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. · The provision of a second assay to corroborate inflammasome activation is consistent with field-specific standards. 2. Quality: Completeness (1–3 scale) SCORE = 2 · Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. · The data in Figs 3/4 could be strengthened by additional assays to measure inflammasome activation beyond IL-1β secretion and PI staining. Options include an IL-18 ELISA, LDH release assay, HMGB1 release assay, or caspase-1 cleavage assay (western blot). · Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. · Beyond additional assays for inflammasome activation, we suggest that the authors could diversify the 'priming' treatments. For example, cells could be primed with LPS, poly I:C, or GM-CSF. Demonstrating CARD8-dependent inflammasome activation in conjunction with diverse stimuli will strengthen conclusions, and take away concerns about a TLR2-specific effect. 3. Quality: Reproducibility (1–3 scale) SCORE = 1 · Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? · Sufficient biological replicates of all assays support the author's conclusions. · Is there sufficient raw data presented to assess the rigor of the analysis? · Yes · Are methods for experimentation and analysis adequately outlined to permit reproducibility? · Yes · If a ''discovery' dataset is used, has a ''validation' cohort been assessed and/or has the issue of false discovery been addressed? · N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5 · Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? · Yes · Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? · Yes · Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions. · The following statement stimulated a lot of discussion amongst the students, who were concerned that the study doesn't really address pathogenesis: "Taken together, our work highlights how even minor, single amino acid changes can have dramatic, species-specific impacts on innate immune sensing and pathogenesis, and provides a model to explain, in part, the unique susceptibility of humans to HIV pathogenesis." · Minor points: We recommend changing the color scheme of graphs in Fig 3 to enhance readability. This could be achieved by color-coding, with one color per group (e.g. HIV-1LAI in blue and HIV-1LAI-VSVG in yellow). MORE SUBJECTIVE CRITERIA (IMPACT): 1. Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE= 2.5 A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] How big of an advance would you consider the findings to be if fully supported but not extended? · If properly supported by corroborating assays, these findings represent a significant advance in the field without further extension. 2. Impact: Extensibility (1–4 or N/A scale) SCORE = 2.5 Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2) · The manuscript explores human vs. chimpanzee CARD8 and diverse lentiviruses. No further extension is provided to a bigger scheme. · We did wonder whether other non-lentiviral proteases could also cleave human CARD8. This would clearly add to our understanding of selective pressures that could have influenced CARD8 evolution in primates. Coronaviruses are mentioned in the discussion. Investigating the activity of coronavirus or enterovirus proteases against CARD8 homologs from different primates would be fascinating. Some clues may already exist in the following n-terminomics studies: o Jagdeo JM, Dufour A, Klein T, Solis N, Kleifeld O, Kizhakkedathu J, Luo H, Overall CM, Jan E. N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection. J Virol. 2018 Mar 28;92(8):e02211-17. doi: 10.1128/JVI.02211-17. PMID: 29437971; PMCID: PMC5874412. o Pablos I, Machado Y, de Jesus HCR, Mohamud Y, Kappelhoff R, Lindskog C, Vlok M, Bell PA, Butler GS, Grin PM, Cao QT, Nguyen JP, Solis N, Abbina S, Rut W, Vederas JC, Szekely L, Szakos A, Drag M, Kizhakkedathu JN, Mossman K, Hirota JA, Jan E, Luo H, Banerjee A, Overall CM. Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome. Cell Rep. 2021 Oct 26;37(4):109892. doi: 10.1016/j.celrep.2021.109892. Epub 2021 Oct 9. PMID: 34672947; PMCID: PMC8501228. o Koudelka T, Boger J, Henkel A, Schönherr R, Krantz S, Fuchs S, Rodríguez E, Redecke L, Tholey A. N-Terminomics for the Identification of In Vitro Substrates and Cleavage Site Specificity of the SARS-CoV-2 Main Protease. Proteomics. 2021 Jan;21(2):e2000246. doi: 10.1002/pmic.202000246. Epub 2020 Nov 17. PMID: 33111431; PMCID: PMC7645863. o Luo SY, Moussa EW, Lopez-Orozco J, Felix-Lopez A, Ishida R, Fayad N, Gomez-Cardona E, Wang H, Wilson JA, Kumar A, Hobman TC, Julien O. Identification of Human Host Substrates of the SARS-CoV-2 Mpro and PLproUsing Subtiligase N-Terminomics. ACS Infect Dis. 2023 Apr 14;9(4):749-761. doi: 10.1021/acsinfecdis.2c00458. Epub 2023 Apr 3. PMID: 37011043; PMCID: PMC10081575. o Bell PA, Overall CM. No Substrate Left behind-Mining of Shotgun Proteomics Datasets Rescues Evidence of Proteolysis by SARS-CoV-2 3CLpro Main Protease. Int J Mol Sci. 2023 May 13;24(10):8723. doi: 10.3390/ijms24108723. PMID: 37240067; PMCID: PMC10218362. Competing interests The author declares that they have no competing interests.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7834266. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Flaviviruses alter endoplasmic reticulum-mitochondria contacts to regulate respiration and apoptosis Wesley Freppel, Anaïs Anton, Zaynab Nouhi, Clément Mazeaud, Claudia Gilbert, Nicolas Tremblay, Viviana Andrea Barragan Torres, Aïssatou Aïcha Sow, Xavier Laulhé, Alain Lamarre, Ian Gaël Rodrigue-Gervais, Andreas Pichlmair, Pietro Scaturro, Laura Hulea, and Laurent Chatel-Chaix.doi: https://doi.org/10.1101/2023.03.09.531853. We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Greater understanding of virus-host interactions are required to support the development of therapeutic interventions for arthropod-borne flaviviruses, Dengue virus (DENV) and Zika virus (ZIKV). Chatel-Chaix and colleagues previously reported that DENV infection alters mitochondrial morphodynamics and that DENV NS4B protein causes elongation of mitochondria, which physically contact ER-derived cytoplasmic substructures called convoluted membranes (CMs) [Chatel-Chaix L, et al. Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses. Cell Host Microbe. 2016 Sep 14;20(3):342-356. doi: 10.1016/j.chom.2016.07.008. Epub 2016 Aug 18. PMID: 27545046; PMCID: PMC7105029]. Mitochondrial elongation compromises the integrity of ER-mitochondria contacts (ERMCs) required to coordinate antiviral responses. In the current study, Freppel, W. et al. investigate flavivirus-induced ERMC perturbations and impacts on processes that involve mitochondria like respiration and apoptosis. They conducted comprehensive ultrastructural analysis of DENV- and ZIKV-infected cells to confirm that infected cells displayed mitochondrial elongation and loss of ERMCs. ERMC loss was measured by proximity ligation assays that monitored the association of three ERMC tethering complexes. Genetic disruption of ERMC contacts by silencing of key ERMC proteins increased viral replication, consistent with the known role of ERMCs in facilitating antiviral responses. Infected cells displayed defects in mitochondrial respiration. Conserved NS4B proteins from ZIKV and DENV were sufficient to recapitulate these mitochondrial defects. Finally, the authors demonstrated that genetically interfering with ERMCs by decreasing levels of key partner proteins suppressed accumulation of executioner caspases in infected cells; accordingly, they conclude that ERMC instability may benefit flaviviruses by apoptosis suppression. OVERALL ASSESSMENT: The authors used a variety of methods to demonstrate ERMC disruption and downstream functional consequences in DENV and ZIKV infected cells. The TEM imaging of mitochondrial morphology and ER-MC interactions, paired with fluorescence microscopy approaches, demonstrated that infection with either ZIKV and DENV alters physical contacts between the mitochondria and the ER and perturbs mitochondrial morphodynamics. Metabolomic, mitoproteomic, and OCR data clearly demonstrated virus-induced mitochondrial stress and diminished respiration, even though mitochondrial membrane potential was maintained. The authors clearly demonstrated that NS4B is sufficient to elicit these changes in ERMCs and mitochondria. STRENGTHS: · The authors built upon past studies of flaviviruses and mitochondria to shine a light on ERMCs as a node of control. · The use of orthogonal approaches to investigate mitochondrial defects was viewed as a strength of the study. This includes the genetic silencing of ERMC tethering complex components which allowed the authors to conclude that mitochondrial function was not only impaired due to morphodynamic change. · We appreciated the use of mitotracker dye to ensure that, despite evidence of mitochondrial stress and diminished respiration, that membrane potential remained intact. · We appreciated the large sample sizes studied during ultrastructural analysis which supported quantitative conclusions. · The demonstration that NS4B proteins from different flaviviruses are sufficient to cause ERMC instability and mitochondrial dysregulation was viewed as a strength of the study. WEAKNESSES: · Uneven writing undermines the impact of this manuscript. The Introduction should be revised for greater clarity and to introduce, and tie together, relevant background and rationale for the study. Some important background information was missing or incompletely described (e.g. NS4B) and there was insufficient introduction of the outstanding questions in the field that are being addressed in this study. Similarly, in the Results section, different experimental approaches are not properly introduced and justified in the context of addressing a research question. One good practice is to begin each new Results section with framing the research question and providing rationale/justification for the experimental approach. For example, such an approach will help the reader appreciate why the authors chose to use mitoproteomics, metabolomics or OCR in different situations to address discrete research questions. · The proximity ligation assays in Fig. 2b would benefit from additional specificity controls, as PLA assays are limited by antibody specificity. · Differences in the timing for observation/harvest should be justified in the text. For example, TEM experiments concluded at 48 hours post-infection (hpi), whereas PLA experiments concluded at 72 hpi. There may be good reasons for these differences, but they should be justified. · The authors nicely paired ZIKV and DENV data throughout the manuscript until Fig. 6, which leaves the reader wondering if DENV also affects apoptosis. Including DENV data in Fig. 6 would provide a pleasing symmetry to the dataset. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2 · Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls. · Fig. 2B: There was consensus that the proximity ligation assays would benefit from additional specificity controls, as PLA assays are limited by antibody specificity. This is required to fully support the claim made in line 171, "This demonstrates a decrease in the abundance of the RRBP1-SYNJ2BP, IP3R1-VDAC1 and VABP-PTPIP51 ERMC tethering complexes and confirms an alteration of ERMCs by DENV and ZIKV at the molecular level." · Fig. 2D: The RRBP1 western blot is poor quality and does not support the claim that RRBP1 decreases over time. Variability in the actin loading control western blot further undermines this claim. Total protein quantitation (e.g. Stain-free technology) provides an attractive alternative to actin. · Are specific analyses performed using methods that are consistent with answering the specific question? · Yes, but as mentioned above, better explanation of the rationale for use of particular experimental approaches would really help the reader. · Is there appropriate technical expertise in the collection and analysis of data presented? · Yes. Clear demonstration of technical expertise throughout. There was only some concern about the quality of western blots in Figure 2. · Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? · Yes. · Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. · Yes. The work is consistent with experimental foundations in the field. 2. Quality: Completeness (1–3 scale) SCORE = 2 · Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. · The authors used a variety of methods to demonstrate ERMC disruption and downstream functional consequences in DENV and ZIKV infected cells. Overall, the use of orthogonal approaches to investigate mitochondrial defects was viewed as a strength of the study. · Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. · No fatal flaws in the paper were identified. 3. Quality: Reproducibility (1–3 scale) SCORE = 2 · Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? · Fig 1: The ultrastructural data shown was quantification from two independent experiments, but it was justified in text that these experimental groups were large enough to obviate the need for a third independent experiment. · Numbers of independent biological replicates (N) should be clearly indicated in all figure legends. · Is there sufficient raw data presented to assess rigor of the analysis? · OK · Are methods for experimentation and analysis adequately outlined to permit reproducibility? · We were concerned about the lack of rationale/justification for choice of experimental approaches to address discrete questions, as well as the variation in time points, sampling procedures, etc. This undermines reproducibility of the research. · If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? · N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5 · Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? Yes · Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? Yes · Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions. · As described above, uneven writing undermines the impact of this manuscript. The Introduction should be revised for greater clarity and to introduce, and tie together, relevant background and rationale for the study. Some important background information was missing or incompletely described (e.g. NS4B) and there was insufficient introduction of the outstanding questions in the field that are being addressed in this study. Similarly, in the Results section, different experimental approaches are not properly introduced and justified in the context of addressing a research question. One good practice is to begin each new Results section with framing the research question and providing rationale/justification for the experimental approach. For example, such an approach will help the reader appreciate why the authors chose to use mitoproteomics, metabolomics or OCR in different situations to address discrete research questions. · Fig 3E: The MitoTracker Orange staining experiments found in the Supplemental Figures establish that membrane potential is largely intact; we think that presenting this data first provides a good foundation for the reader before delving into the mitochondrial respiration measurements in Fig. 3E. · Minor comments: · Fig 1C: bar graphs would be easier to interpret if brackets marked the groups being compared for statistical significance. · Fig 2A: The text in this cartoon is small and difficult to read. · Fig 3F: The heat map data superimposed on a Krebs Cycle/ETC cartoon is complicated and hard to interpret. We suggest that a clearer representation of the heat map alone will really help the reader interpret this data better, whereas the cartoon could be displayed for reference elsewhere. · Numbers of independent biological replicates (N) should be clearly indicated in all figure legends. MORE SUBJECTIVE CRITERIA (IMPACT) 1. Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE= 1.5 A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] · This manuscript builds upon a very impactful previous paper from Dr. Chatel-Chaix et al. in Cell Host & Microbe in 2016. In a sense, the demonstration of ERMC disruption and mitochondrial dysfunction in cells infected with DENV or ZIKV provides evidence for a broader importance of these discoveries. This manuscript provides a fundamental foundation for understanding this node of control in virus infected cells, which could be applicable for diverse viruses beyond these two flaviviruses. Impact: Extensibility (1–4 or N/A scale) SCORE = 1.5 · Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? · No extension to human cohort necessary for this to represent an important advance. · This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2) · N/A Competing interests The author declares that they have no competing interests.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7737272. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories Jingru Fang, Guillaume Castillon, Sebastien Phan, Sara McArdle, Chitra Hariharan, Mark H. Ellisman, Ashok A. Deniz, Erica Ollmann Saphire. doi: https://doi.org/10.1101/2022.12.27.522024 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Many fundamental aspects of Ebola virus (EBOV) replication remain unknown. Here, the authors sought to better understand the role of EBOV viral factories (VF) in spatially regulating viral RNA synthesis. They reconstituted VFs using viral proteins with properties often associated with phase separation (self-oligomerization, RNA binding) including nucleoprotein (NP), polymerase cofactor VP35, and polymerase (L). Condensates were observed in cells transfected with VP35 alone, VP35/NP, or VP35/NP/L. These condensates displayed composition-dependent viscoelastic behavior typical of VFs. Interestingly, L was found to cluster in foci that were interconnected but the distance between the foci was dependent on RNA replication (provided by a minigenome (MG) reporter). VFs formed a typical droplet-like morphology as well as a distinct network-like morphology. To determine if both VF morphologies were present during infection, the authors performed infections with a VP30-deficient EBOV in Vero cells that provide VP30 in trans. They determined that most VFs display droplet-like morphology, but network-like morphology is still present about 30% of the time. Continuing with their transfection-based reconstitution system, the authors designed a split APEX2 (sAPEX2) system to monitor interactions between L and VP35. TEM confirmed the network-like morphology of VFs and localization of L at the periphery of these structures. Finally, using the sAPEX2 system, they utilized four-tilt electron tomography (ET) to resolve 3D images of organelles. Using ET, they obtained clear resolution of the reconstituted VFs and observed that they were close to membrane bound organelles, consistent with previous observations of VFs from other viruses. Interestingly, they also reported loosely coiled structures which they proposed are viral ribonucleoproteins, however this requires further investigation. Together, Fang J., et al. demonstrated the utility of their transfection-based approach for studying EBOV and advanced understanding of EBOV VFs, describing a network-like morphology and composition-dependent distribution of L within VFs. OVERALL ASSESSMENT: This preprint provides advances understanding of EBOV VFs as biomolecular condensates, providing insight into the interactions, viscoelastic properties, and spatial organization of VF constituents. The study featured cutting-edge microscopy techniques. This study could be strengthened by the addition of key controls, especially for the sAPEX2 system. We also suggest adjustments to the writing to better highlight important findings. STRENGTHS: This study addresses important questions about the fundamental nature of VFs. The writing was generally clear and accessible, with clear communication of research goals and approaches throughout. For example, the clear Introduction and accompanying methods diagram was very helpful for readers with limited virology knowledge. Without question, the advanced microscopy approaches are impressive, and provide a roadmap for future studies. We appreciated the authors cautious assessment of their findings, which acknowledged experimental limitations and did not overinterpret results. Overall, conclusions were well supported by the data throughout. WEAKNESSES: We identified the following weaknesses: 1. While we appreciated the power of the sAPEX2 assay to map localization of protein complexes, we think that it could be improved by adding specificity controls. The specificity of the mapping of L+VP35 could be better appreciated if it could be compared to controls (e.g. viral proteins that lack condensate properties and do not participate in VFs). 2. The authors should double-check figure callouts throughout the paper as there are several occasions where the figure they are referring to does not relate to the claim in the sentence. This is a minor weakness, as the data is usually somewhere in the paper, but the misleading callouts were frustrating. 3. Another minor weakness involved the descriptive terminology used in the paper such as "gel-like" or "homogeneous". The authors should define these terms clearly so the reader can understand how they are being used in the context of this study. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.25 ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls. ● Figures are generally attractive and informative, and the data supports the authors' conclusions. Relatively minor critiques as follows: o Figure 1: The figure legend has an error, as the descriptors for A and B are reversed. We encourage the authors to review figure callouts in the text as, as there are mismatches. They also refer to Figure 1e (line 165) and it is unclear how that figure supports the claim in the text. We think it would be more appropriate to reference Supplemental Figure 1d along with Figure 1e to support the claim. o Figure 2: To improve the continuity of Figure 2C, we suggest the authors improve the alignment of the x-axis of the graph with the lanes of the corresponding western blot below. We suggest straight labels or dotted lines to clearly indicate the continuity from the graph to the lanes of the blot. We also suggest increasing the signal of the anti-FLAG blots to give a more obvious positive signal and reveal any additional bands that are currently obscured. o Figure 3: We suggest clearly indicating which panel shows the network-like or droplet-like phenotypes in Figure 3b. We propose direct labeling on the figure or referencing left and right panels in the figure legend. o Figure 4: It is unclear what "1" and "2" refer to in the labeling of Figure 4d; we recommend clarifying this on the figure or in the figure legend. It would also be beneficial to include Z-stacks for Figures 4e and 4f to allow the reader to better locate L-VP35-sAPEX2. We also identified a significant error in the interpretation of the data in Figure 4b and 4d. In the text it reads "L-sEX was also expressed to higher levels than wild type L, which could explain the correspondingly higher MG activity seen for sAPEX2-tagged EBOV polymerase (Figure 4d)" however, Figure 4d only shows the expression levels. Furthermore, we do not understand this claim as the MG activity shown in Figure 4b revealed that L-VP35-sAPEX2 had lower activity compared to L-WT + VP35-V5. We encourage the authors to revise this section of text. o Figure 5: For consistency, a scale bar should be included in the OsO4 panel of Figure 5b. Our more substantial concern is the lack of controls for the sAPEX2 assay. While we acknowledge that this is an innovative tool, it would be more convincing to see the labeling of something that is not their target, also in the phase condensate, which has a different distribution. We suggest potentially using the sAPEX2 assay with NP and VP34. The use of this control would better validate their assay since their observations of the network-like structures on the periphery of VFs seem to contradict the IF data from figure 4. ● Are specific analyses performed using methods that are consistent with answering the specific question? o We have concerns about the use of the mini-genome as the substrate for polymerase L and indicator of viral replication and transcription within VFs/condensates. Since condensate formation is heavily impacted by the stoichiometry of its constituents, we are concerned that the use of this mini-genome may influence phase separation more than the authors have anticipated. We would also like the authors to discuss the potential implications of the higher expression of VP35 APEX compared to the VP35-V5 construct. Finally, we suggest that the authors use RNA FISH staining to visualize viral RNA to further enhance confocal microscopy experiments. . ● Is there appropriate technical expertise in the collection and analysis of data presented? o The sAPEX2 construct impacted the intracellular localization pattern of VP35 and induced the formation of network-like VFs. Although the authors showed that co-expression of VP24 and VP35-V5 could also induce the network-like VFs, there are still concerns about potential artefacts caused by sAPEX2 tags. While potentially beyond the scope of this excellent paper, we suggest the authors consider supporting their data with alternative labeling techniques such as immunogold labeling or metal-tagging. The immunogold labeling and metal-tagging have their own limitations, but those techniques do not create extra intermolecular interactions between L and VP35, therefore eliminating potentially artefactual effects on VF morphology. Based on the information in the Results section, the sAPEX2 tagging only allows the authors to examine the network-like VFs with TEM but not droplet-like VFs. It would be very interesting to visualize the droplet-like VPs and the corresponding VP35 distribution. Would the distribution of the VP35 in two phases of VFs align with the IF data? What is the structural/component differences between the network-like and droplet-like VFs? This may provide additional evidence to support the proposed model in the Discussion. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o OK. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o OK. 2. Quality: Completeness (1–3 scale) SCORE = 1 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o OK. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o N/A. 3. Quality: Reproducibility (1–3 scale) SCORE = 1.25 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o While we recognize generating the confocal microscopy data is a cumbersome task, we would like to see three biological replicates for the data presented in Figure 3. ● Is there sufficient raw data presented to assess rigor of the analysis? o OK. ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? o Excellent throughout. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o OK. ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o Concepts first covered in the Introduction should be revisited in the Discussion to "close-the-loop" and make for more satisfying reading. Please include citations when discussing knowledge gaps and cite other studies that demonstrate why it is important to address these knowledge gaps. Without this, the reader is left with little to refer to other than the Introduction. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions. o We recommend the authors use summary statements as headings in the Results, which would aid reader comprehension throughout. Since the authors created useful methodology diagrams, we suggest the inclusion of a diagram for their proposed model, even though some aspects of the model remain speculative. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.5 A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o The novelty of this study is two-fold; the findings address key knowledge gaps and contribute to a better understanding of Ebola virus VFs, and the validation of the transfection-based minimal system for studying VF condensate properties provides a valuable guide for others. Using the transfection-based model, they observed the contribution of individual viral constituents to VFs and condensate formation. Specifically, they found a reduction in the molecular exchange rate in VFs composed of NP and VP35 in the presence of L. Additional observations included the potential for NP to act as a scaffold in VFs and the influence of viral RNA synthesis on polymerase localization. The authors suggest that these discoveries can be applied to other non-segmented, negative sense RNA viruses. These impressive discoveries might not be fully appreciated by the non-expert reader without further exposition. As suggested in the "scholarship" critique above, revisiting and reinforcing key concepts and providing impact statements in the Discussion may help in this regard. Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? N/A ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). N/A Competing interests The author declares that they have no competing interests.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7675558. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Distinct pathways of adaptive evolution in Cryptococcus neoformans reveal a point mutation in adenylate cyclase with drastic tradeoffs for pathogenicity Zoë A. Hilbert1,3*, Krystal Y. Chung2, Joseph M. Bednarek2, Mara W. Schwiesow1,3,4, Jessica C.S. Brown2, Nels C. Elde1,3* doi: https://doi.org/10.1101/2022.09.27.509772 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: The genetic diversity of pathogenic microorganisms is shaped by host-pathogen interactions. Rapid adaptation of viral and bacterial pathogens to new hosts is well documented but studies of environmentally derived pathogenic fungi are limited. Our understanding on how host-fungi interaction affects pathogenesis remains unclear. Here, Hilbert et al., investigated how the evolution of the environmentally derived pathogen Cryptococcus neoformans has been shaped by interactions with host cells. The authors evaluated the ability of 14 clinical and environmental isolates of C. neoformans to replicate in amoebae (Acanthamoeba castellanii) or J774A.1 cell lines (macrophage-like cells). Notably, the environmental isolate Ftc555-1 could replicate in macrophages but could not survive in cell culture media alone. The authors leveraged this phenotype to study the evolution of C. neoformans by host selective pressures in the absence of extracellular fungal growth. They performed serial in vitro passages of the C. neoformans Ftc555-1 strain in either amoeba or macrophages for a total of 12 passages to generate the adapted strains for each type of host cell. The authors noted that one of the adapted strains, known as M1, that was recovered from the serial passaging in macrophages showed a strong host-adapted phenotype with 5-fold increase in replication in macrophages compared to its parental strain. Whole genome sequencing of the M1 strain revealed that only a single nucleotide polymorphism in the adenylate cyclase gene CAC1 was acquired and swept to fixation in the final passage of the experimental evolution, which resulted in an Arg1227Pro substitution in the enzyme. The authors established that this substitution was sufficient for enhanced intracellular growth of M1. The authors showed that swapping the CAC1 allele in the parental Ftc555-1 strain with the mutated CAC1 allele (cac1-evo) from M1 strain was sufficient to induce M1-like phenotype. In addition, swapping the cac1-evo allele in the M1 strain with the parental CAC1 allele caused a complete loss of the M1 phenotype. Whole genome sequencing of intermediate stages in experimental evolution, known as the "fossil record", showed that the cac1-evo allele arose very early during the serial passage and quickly spread through the population, which correlated with phenotypic changes in fungal growth in macrophages. This suggests that the cac1-evo allele confers a strong fitness advantage. Interestingly, the authors found that the Arg1227Pro substitution limited capsule formation by the M1 strain or cac1-evo allele-swapped Ftc555-1 strain. Finally, the authors demonstrated that the M1 strain failed to cause lethal disease in a mouse infection model, while the parental Ftc555-1 strain had a median time to endpoint of 34 days. This suggests a tradeoff between the in vivopathogenicity of M1 strain and its enhanced fitness in macrophages in vitro. OVERALL ASSESSMENT: Experimental evolution studies often provide insights into host-pathogen interactions. Here, Hilbert et al, performed in vitro serial passaging of C. neoformans in different hosts to learn about fundamental mechanisms of host adaptation. The authors demonstrated that a non-synonymous mutation in CAC1 was sufficient to provide the M1 strain with an in vitro intracellular growth advantage in macrophages. The CAC1 mutation also affected fungal capsule thickness, which is known to be regulated through cAMP pathway. Overall, we considered the experimental evolution analysis of this study to be impressive and well structured. The main finding of this paper provided important insights into growth regulation of C. neoformans and highlighted the importance of cAMP pathway in fungal biogenesis. In our discussions, there was general agreement that the authors could improve the impact of their work by including mechanistic studies about the CAC1 mutation identified in the M1 strain. The final part of the study focused on characterization of the in vivo pathogenicity of the M1 strain. We were intrigued by the increase in vitrogrowth that did not correlate with lethality in mice. However, we were concerned about the completeness of this aspect of the study and encourage further exploration. STRENGTHS: The main strength of the paper is the demonstration of rapid host adaptation of an environmental isolate of C. neoformans to a new host. This finding contributes to our understanding of the microevolution events of pathogenic fungi, which deserves more attention in the field. The strong correlation of the intracellular survival phenotype of the adapted M1 strain to the single amino acid substitution on CAC1 protein highlights the importance of the cAMP signaling pathway in controlling the survival and pathogenesis of C. neoformans. WEAKNESSES: We identified the following weaknesses: 1. The experimental evaluation in this paper resulted in a C. neoformans strain with a strong intracellular growth advantage. The authors pinpointed the specific mutation that is responsible for the strong phenotype but did not extend their work into studies of the molecular mechanisms that drive the phenotype. 2. Some aspects of data presentation were incomplete. For example, microscopy images of the fungal capsule were not included in the paper, even though it would have helped the reader (only quantitative data shown). Furthermore, the animal data was incomplete, as the authors could have shared data like body weight changes and appearance that are routinely collected during mouse studies to help bolster their conclusions about pathogenesis. 3. Based on the information given in the Introduction, the amoeba is a potential source of environmental selective pressure for virulence factors of microorganisms. This is due to the similarities between amoeba and macrophages. It justifies the use of amoebae in this paper as the environmental host for C. neoformans. However, the amoeba studies were underdeveloped and would benefit from further mechanistic elaboration. Furthermore, the fact that the macrophage adapted strains did not show growth advantage in amoeba also did not align with claims made in the Introduction. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2 ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls. The general comment we have about the figures is the way that statistical significance was presented. We found that indicating significance (* or NS) on top of the bar did not clearly indicate which two values were being compared. For example, in Figure 1B, it was unclear if each value was compared to H99O or compared to the neighboring value. In addition, it was unclear if the significance (* or NS) on top of each strain reflected values of both media and co-culture groups or for only one of these groups. We suggest that the statistical analysis could be presented more clearly. Figure 3: This figure provides key supporting data to validate that the intracellular growth is being selected and improved in the M1 strain. We think that the fluconazole data in Supplemental Figure 3 provides important support and that the reader would benefit from including it as part of Figure 3. Figure 5: Here, the authors comment on the "striking difference in the pathogenicity of the serially passaged M1 strain when compared to the parental Ftc555-1 strain". To support this statement, the authors should compare the level of fungal infection in mice between the M1 strain and the parental Ftc555-1 strain. We really think it is important to include the CFU in the lung and brain of the mice infected with Ftc555-1 parental strain as a critical control, which also serves as a base line of the C. neoformans infection in mice. ● Are specific analyses performed using methods that are consistent with answering the specific question? The difference of the survival rate between the mice infected with M1 and parental strain was striking. It is indeed a strong implication for a reduced pathogenicity of the M1 strain. In our opinion, there are many inter-related criteria that affects the pathogenicity of any given pathogenic microorganisms, such as their ability to cause disease, the severity of the disease, their ability to establish an infection, and their ability to replicate in the host. We appreciate the discussion of those criteria in the paper, but we believe more data from the animal infection experiment is required to give better support of the pathogenicity difference between M1 and parental strain. We think it would be helpful to show the mouse body weight that was measured during the infection. Especially, it would be interesting to see how the body weight changes during the first 20 days post infection. We understand that the in vivo study is expensive and time consuming, but growth conditions are known to affect the capsule. Thus, we think that if the authors could perform a capsule assay post-infection it will forge a stronger link between the in vitro and in vivophenotypes of M1. It would also be interesting to measure the intracellular levels of M1 and the parental strain in the liver macrophages of the infected mice, as the liver macrophages are known to play an important role in C. neoformans infection. ● Is there appropriate technical expertise in the collection and analysis of data presented? Yes. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? We think that the one-way ANOVA tests used throughout are generally acceptable. However, we would like to address our concerns about the stringency of using one-way ANOVA with multiple comparison in Figure 1C, which only has two biological replicates. We suggest either using t-test to compare the growth difference between each isolated strain to the control strain or completing the dataset with third biological replicate, to enable ANOVA analysis. In addition, we were confused by the use of both t-tests and ANOVA in Figure 5A. It created confusion about the significance of the results by using two different statistical tests with overlapping functions for the same dataset. The comparison between the M1 and FTc555-1 cac1 mutant strain can be done with multiple comparison tests that normally run alongside of one-way ANOVA. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. N/A Adequate. 2. Quality: Completeness (1–3 scale) SCORE =1.5 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. We think the title and abstract are generally well supported by the data presented in the paper. However, we want to address our concerns about the animal infection data that was used to support the reduced pathogenicity of M1 strain as a tradeoff for its intracellular growth advantage in vitro. As mentioned in the previous section of this review, only showing the mice survival data is not sufficient to establish the conclusion about pathogenicity of the M1 strain. Data from the parental strain is needed in all assays to show the base line of the C. neoformans infection. Other aspects of the infection should also be evaluated, such as the animal weight changes, capsule thickness of both strains post-infection, and the growth of both strains in infected mouse macrophages. It is also important to have biological replications for the animal experiments to show the stringency of the results. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. N/A 3. Quality: Reproducibility (1–3 scale) SCORE = 2 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? Most experiments have at least 3 biological replicates, with two exceptions. 1. The experiment testing the replication of C. neoformans strains in co-incubation experiments with amoeba cells (Figure 1C) only has two biological replicates. We believe either including a few sentences to justify the reason for having only two replicates or adding an extra biological replicate of the experiment would increase the stringency of the results. 2. According to the methods, the C. neoformans mice infection has only been done once. We believe that at least three biological replicates are required for the mice infection model to show the reproducibility of the results. In this way, the results of Figure 5C will better support the "tradeoff" phenotype of M1 strain, as the authors concluded. If there were more than one biological replicate for the animal experiments, we suggest including the information clearly in the Methods and Figure caption. ● Is there sufficient raw data presented to assess rigor of the analysis? The raw data provided is generally sufficient. However, we found that only violin plots were included in the paper to show the effects of CAC1 mutation on the capsule thickness of C. neoformans. We suggest including the microscopy images that the measurements were performed on in addition to the violin plot. We believe including the images will help the audience to better visualize the difference of the capsule size/thickness between different strains. This will provide better support for the statement "the point mutation in CAC1 gene affects the C. neoformans capsule production". ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? Yes. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE =1.5 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? OK ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? OK ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions. We think there are few points in the paper that could be improved by making small changes. This includes the following: 1. In Figure 3, the legend for the solid and striped bar was missing. There was typo in the figure caption for Figure 3A "Solid barsindicate competitive indices in media only, solid bars indicate competitive indices in the macrophage". 2. We think that including cAMP pathway diagram in Figure 4A raised questions about the mechanisms of how cac1 mutation affected the cAMP pathway, which was not investigated in depth the manuscript. Therefore, we suggest removing Figure 4A if cAMP pathway genetic and physical interactions are not going to be studied in detail. 3. We were not sure the exact massage the authors want to deliver with the sweeping statement [Regardless of exact mechanism, the complex impact of a single nucleotide change in the genome of this pathogenic fungus highlights the long reach of simple adaptive changes to fundamentally change the course of evolution]. We were specifically confused about the meaning of "fundamentally change the course of evolution". We suggest that expanding this statement could help the audience understand how the preprint supports these conclusions. 4. We noticed that the y-axis scale of Figure 5A only has 0, 5 and 10, which makes it

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7635763. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses Jing Wang1, Yuan-fei Pan2, Li-fen Yang3, Wei-hong Yang3, Chu-ming Luo4, Juan Wang3, Guo-peng Kuang3, Wei-chen Wu1, Qin-yu Gou1, Gen-yang Xin1, Bo Li5, Huan-le Luo4, Yao-qing Chen4, Yue-long Shu4, Deyin Guo1,6, Zi-Hou Gao3, Guodong Liang7, Jun Li8, Edward C. Holmes9*, Yun Feng3*, Mang Shi1* doi: https://doi.org/10.1101/2022.11.23.517609 We adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Bats are reservoir hosts for emerging and re-emerging viruses of concern, including influenza viruses and coronaviruses with pandemic potential. Understanding the natural history of these emerging viruses in their reservoir hosts is a top priority for global surveillance efforts. In this manuscript, Wang, J., et al. study the viromes of individual bats from different species collected in Yunnan province to start to understand how viruses of concern co-infect certain bats and/or transmit between species, and to attempt to identify viruses with characteristics that could support zoonotic transmission to humans. This focus on individual bat viromes improves upon previous studies that pooled data from bats of the same species. The authors collected rectal samples from 149 bats from 15 different species from six areas in Yunnan province and harvested RNA for metatranscriptomic sequencing. They focused on viruses that infect mammals. Initial characterization of viromes showed the most frequent viral families across all bat species were Reoviridae, Picornaviridae and Coronaviridae. One third of bats were co-infected, but co-infection rates differed between bat species. Relatively few virus species were detected in more than two bat species. Using a Poisson regression model, they showed that there is a significant correlation between virome composition and the physical proximity and phylogenetic relatedness of bats. To tie their research into potential viral emergence they first identified viruses that are closely related to known human or livestock pathogens. Of these identified "viruses of concern" they focused on several coronaviruses, two of which (BtSY1 and BtSY2) were closely related to SARS-CoVs based upon similarity in RdRp sequences. To further characterize BtSY1 and BtSY2 they compared the sequences of several genes (RdRp, NTD, RBD of spike and N) to other related coronaviruses. From this analysis they showed BtSY1 clusters with the "S-1" clade of coronaviruses (like SARS-CoV) and BtSY2 clusters with the "S-2" clade (like SARS-CoV-2). Comparison of whole genome sequences demonstrated that BtSY1 is 93% identical to human SARS-CoV viruses and BtSY2 is 92% identical to SARS-CoV-2. They also provided evidence for recent recombination events in the origin of BtSY2. Finally, using structural homology-modeling of BtSY2 RBD they hypothesize BtSY2 can use human ACE2 receptor to enter cells like SARS-CoV-2. Overall, these studies suggest that transmission of viruses between different bat species can lead to co-infection of individuals required for recombination events, analogous to the "antigenic shift" events that cause periodic emergence of pandemic influenza viruses. OVERALL ASSESSMENT: This preprint breaks new ground in the study of viromes in reservoir hosts for the purpose of pandemic surveillance. Study of viromes in individual bats from diverse, geographically linked species were required to get a clear assessment of potential transmission, co-infection and recombination events. Importantly, this approach enabled the discovery of two novel bat viruses closely related to SARS-CoV and SARS-CoV-2, which demonstrates the value of the approach. Comparative genomic analysis revealed recombination events in the origin of BtSY2. STRENGTHS: A clear strength of this study is the novel approach of investigating viromes of individual bats from different geographically linked species, which allowed the authors to investigate co-infection and potential recombination events. This provides a roadmap for the identification of novel viruses, and virus-host combinations, that increase risk of spillover to humans. The conclusions are generally well supported throughout, and the identification of potential viruses of concern closely related to SARS-CoV and SARS-CoV-2 is undoubtedly very important and will spur future studies. WEAKNESSES: The primary weaknesses of the study relate to communication. The impact of this study is clear but could be further enhanced by improvements in data presentation and expository writing. We think that it could be more impactful if split into two 'back-to-back' manuscripts, with the first focused on the metatranscriptomic studies and cataloguing diverse new viruses discovered and their potential transmission between geographically linked hosts from different bat species, and the second focused on the two novel sarbecoviruses of concern and their properties. This would give the different components of this impactful study the attention they deserve. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.5 · Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] · Figure 2: We found it challenging and overwhelming to interpret the wealth of information presented in this figure. The top of the heat map has the samples grouped by three different parameters (host genus, host species, and sample location). Potentially limiting it to a single parameter along each axis would aid in making comparisons across the parameters easier. · Figure 4A: We were uncertain about what branch length represents in this figure. Please clarify. Could the green circles representing "other viruses" be condensed to a single circle with the number of viruses it represents, or omitted, since the viruses are not named and not central to the message of the figure? · Figure 5: We were overwhelmed by the amount of information in this figure. This could be improved by limiting the figure to display the tree of one or two viral families that include the coronaviruses that form the focus for the remainder of the preprint. The other trees could move to the data supplement. · Figure 6B: The recombination analysis is fascinating, but underdeveloped, and left us wanting more. The reader would benefit from higher resolution information about proposed sites of recombination (maybe provided by insets?) and how they relate to known recombination hot spots. Undoubtedly, informative comparisons could be made between other related viral genomes. To help the reader make comparisons, colour schemes should be harmonized across the different plots. · Are specific analyses performed using methods that are consistent with answering the specific question? · Generally, this was viewed as acceptable throughout. · Is there appropriate technical expertise in the collection and analysis of data presented? · Generally, this was viewed as acceptable throughout. · Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? · Generally, this was viewed as acceptable throughout. · Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. · Was the health of the animals assessed? We discussed how findings might differ between healthy and sick bats, and if this would be reflected in viral load or diversity. · Knowing that bat trapping and sampling was conducted over time made us wonder about how that might affect the genotypes of viruses circulating in these populations over different years. Was this information collected? Is there any evidence that viromes changed over time in certain bat species in the study? 2. Quality: Completeness (1–3 scale) SCORE = 2 · Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. · The early exclusion of non-mammalian viruses should be justified. · Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. · Generally, this was viewed as acceptable throughout. 3. Quality: Reproducibility (1–3 scale) SCORE = 1.5 · Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? · N/A · Is there sufficient raw data presented to assess rigor of the analysis? · Generally, this was viewed as acceptable throughout. · Are methods for experimentation and analysis adequately outlined to permit reproducibility? · Generally acceptable. We thought that providing a clearer rationale for narrowing focus to mammalian viruses, and then the novel sarbecoviruses, would be helpful. · If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? · N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 2 · Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? · Generally, this was viewed as acceptable throughout. · Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? · Generally, this was viewed as acceptable throughout. · Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant basis for decisions. · The impact of this study is clear but could be further enhanced by improvements in data presentation and expository writing. · We think that it could be more impactful if split into two 'back-to-back' manuscripts, with the first focused on the metatranscriptomic studies and cataloguing diverse new viruses discovered and their potential transmission between geographically linked hosts from different bat species, and the second focused on the two novel sarbecoviruses of concern and their properties. This would give the different components of this impactful study the attention they deserve. MORE SUBJECTIVE CRITERIA (IMPACT) 1. Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1 A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] · How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. · The work already represents a substantial advance. A clear strength of this paper is the roadmap to characterizing the viromes of individual reservoir hosts, and the demonstration of co-infection events that provide opportunities for recombination and emergence of novel viruses. Beyond this, the identification and partial characterization of the two novel sarbecoviruses, BtSY1 and BtSY2, represents a very important finding, along with evidence for recombination in the genesis of these viruses. 2. Impact: Extensibility (1–4 or N/A scale) SCORE = 2.5 · Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? · There are certainly opportunities to extend these studies to experimental infection models, but this exceeds the reasonable scope of this research. However, we were keen to extend the studies of BtSY2 RBD binding to ACE2, which were limited to in silicoanalyses. Synthesis of BtSY2 Spike protein and testing directly in ACE2 binding assays is quite feasible and straightforward, as is the creation of BtSY2 pseudoviruses that could be used to test virus entry mechanisms and neutralization by animal sera. We view such experiments as achievable and well within the scope of the current study. · This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). N/A Competing interests The author declares that they have no competing interests.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7585120. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: ISG15-modification of the Arp2/3 complex restricts pathogen spread Yifeng Zhang1, Brittany Ripley1, Wei Ouyang2,3, Miranda Sturtz1, Ellen Upton1, Emma Luhmann1, Madeleine Vessely1, Rocio Coloma4, Nathan Schwery1, Scott M. Anthony5, Adam 5, Thomas O. Moninger6, John T. Harty5, Aloysius Klingelhutz1, Emma Lundberg2,3,7, 5, Balaji Manicassamy1, Christopher Stipp8, Susana Guerra4, and Lilliana Radoshevich1* doi: https://doi.org/10.1101/2022.12.27.522022 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: ISG15 is a Ubiquitin Like (UbL) protein that is linked to antimicrobial defense. Like all UbL proteins, ISG15 can be covalently linked to substrate proteins, a process known as ISGylation. ISGylation is intimately linked to interferon (IFN) responses; in fact, IFN responses drive transcriptional activation of the genes that encode the ISGylation machinery, including ISG15 itself. As ISG15 substrate proteins are identified, understanding of how ISGylation affects the fate of substrate proteins and relevance to antimicrobial defense must be addressed. Here, Zhang, Y., et al. build upon their previous comprehensive proteomics studies that identified ISGylated proteins in Listeria monocytogenes (Lm) infected animals (REF: Zhang Y., et al (2019) Nat. Commun. 10, 5383. Doi: 10.1038/s41467-019-13393-x). They employed a very clever approach that compared infected WT, ISG15 KO and USP18 C61A animals. In particular, the USP18 mutant animals displayed hyper-ISGylation due to the defective ISG15-specific USP18 deconjugase, which allowed the authors to distinguish between ubiquitin, ISG15 and the NEDD4 UbL modifications, all of which yield a di-glycine dipeptide on tryptic peptides during processing for mass spectrometry. Here, the authors report that hyper-ISGylation paradoxically increases Lm lethality in mice and increases pathogen spread in vivo and in vitro. They hypothesized that if hyper-ISGylation aids Lm cell to cell spread, which depends on the propulsive force of actin tails, then tail components may be substrates. They cross-referenced their list of candidate ISGylated proteins from Lm-infected mice with a list of host proteins known to be associated with Lm actin tails and discovered ISGylation of several proteins from the actin branching Arp2/3 complex, including Arp2 and Arp3 themselves. They demonstrated that ISG15 is present in Lm actin tails, and that hyper-ISGylation was associated with altered Arp3 localization on the Lm cell surface (rather than being restricted to the tail). Hyper-ISGylation was also associated with shorter tails and decreased Lm motility. Knowing that Arp3 is a substrate, they modelled ISGylation on 3 substrate lysines, which suggested the possibility of decreased Arp2/3 complex activity through steric hinderance. In the hyper-ISGylation cells, they noticed a phenomenon of groups of bacteria, which they called 'bazookas', invading neighboring cells. Careful time-lapse microscopy analysis suggested a failure of daughter bacteria to separate post-division in the hyper-ISGylation cells, which the authors conclude is the likely cause of the grouping phenomenon. They were able to link this phenomenon back to Arp2/3 complex ISGylation using USP18 C61A cells in which Arp3 was knocked out and complemented with a non-ISGylatable mutant Arp3 construct (with the 3 key lysines substituted); in these cells, hyper-ISGylation was no longer able to cause the bacterial segregation defect. Finally, to link these studies to broader control of cell biology and development by ISGylation of Arp2/3 complex proteins, the authors determined that hyper-ISGylation causes the accumulation of thick cortical actin in fibroblasts, stronger attachment to a fibronectin matrix, slower cell migration, and general thickening of epithelial layers in mice. OVERALL ASSESSMENT: Discovering new UbL modifications of substrate proteins and determining the effects of these modifications on protein fate are difficult tasks. In this manuscript, Zhang Y. et al., demonstrate that Arp2/3 complex proteins are ISGylated and that perturbations in ISGylation affect the actin tails that propel Lm. Conclusions are generally well supported by the data throughout, particularly with regard to the Lmmotility data. The authors employ advanced microscopy to demonstrate that increased global ISGylation causes defects in actin tails that support greater cell-to-cell spread of bacteria via a clustering effect. In particular, the role of Arp3 ISGylation in driving these defects is well supported through the study of mutant alleles of Arp3 that cannot be ISGylated. Overall, we considered this study to be an impressive contribution to the understanding of how UbLs can affect host-pathogen interactions. By contrast, we considered the final figure that focused on general effects of ISGylation on fibroblast actin cytoskeleton dynamics, cell shape, motility and epithelial barrier function, to be less well developed and somewhat disconnected from the strong host-pathogen interactions work that preceded it. STRENGTHS: The novelty of this paper is a strength, and larger conclusions are supported by the data. The authors provide new mechanistic insight into how ISGylation affects host-pathogen interactions by modifying the Arp2/3 actin branching complex, which we considered a big advance considering our generally poor understanding of ISGylation to date. This also suggests that the proteomic dataset from their Nature Communications paper may contain more valuable ISGylated host proteins that could be the subject of future studies. WEAKNESSES: We identified weaknesses that we would like to address: 1. There was broad agreement that while the alterations in fibroblasts and epithelial barrier in the hyper-ISGylated state (Fig. 5) are fascinating, this aspect of the study is less well developed compared to the host-pathogen studies that preceded it. For example, while there were clearly changes in the fibroblast actin cytoskeleton, shape and motility that could be plausibly linked to ISGylation of the Arp2/3 complex and global defects in actin branching, only the motility phenotype was clearly linked to Arp2/3 complex, and this information was relegated to the extended data. Even if additional confirmatory experiments were performed to shore up this aspect of the manuscript, it would remain conceptually peripheral to the main take-home message. We generally agreed that the manuscript would be stronger without Figure 5. 2. There were some statements in the manuscript that were not well supported and should be reconsidered. This includes the following: a. Title: "ISG15-modification of the Arp2/3 complex restricts pathogen spread" - this is confusing as the main message is that aberrant hyper-ISGylation of Arp2/3 results in actin tail alterations (shorter, thicker) and failed bacterial segregation post-division, which nevertheless increases spread due to the formation of bazookas. In this light, 'restriction' doesn't seem like the right description of the change. It is certainly true that the ISG15 KO cells display an enhanced bacterial spread phenotype, but we didn't think this was the main take-home message. Also, the use of '"pathogen" is meant to include VACV, but there was not very much virus infection data in the manuscript to support this broader scope of the title. b. The flow of information in the abstract does not match the order of figures. It doesn't have to, but if the general effects of hyper-ISGylation on cortical actin and lamella need to be in the 3rd sentence of the abstract, then maybe the data in Figure 5 belongs in the first figure. c. The final sentence of the abstract is an impact statement that goes beyond the scope of the study. "Ultimately, our discovery links host innate immune responses to cytoskeletal dynamics with therapeutic implications for viral infection and metastasis." The authors should consider revising this statement to keep focus on the main findings of the study, as the therapeutic options remain uncertain, and the main point of this compelling study is deepening understanding of host-pathogen interactions, not cancer. 3. The experiments involving the Arp3 KO cells complemented with the non-ISGylatable mutant Arp3 allele provide essential mechanistic information linking phenotypes to the Arp2/3 complex and steering the reader away from potential indirect effects of ISGylation on cell physiology. As such, we think that the data in Extended Data Figure 4 should be retrieved and put in the main dataset. 4. We know from Extended Data 3b/c that the ActA-R148S mutant Lm bacteria that could not stably recruit Arp3 lost its enhanced cell-to-cell spread phenotype in hyper-ISGylation cells. This clearly indicates that ISGylation of Arp2/3 complex components AND their recruitment to bacteria is required for this phenotype. However, there is much more to learn from the ActA-R148S mutant Lm bacteria. For example, how do we expect these mutant bacteria to perform in vivo, in WT or USP18C61A/C61A mice? Do the mutant bacteria still replicate and spread efficiently in the liver, and does the hyper-ISGylated state have any effect? 5. We found the intravital microscopy data in Figure 1 challenging; there appear to be changes, but they are difficult for the non-expert to interpret properly. We think that retrieving the 2-photon microscopy images from the Extended Data could aid interpretation. 6. Infectious doses, timings, and cell types for infection assays varied throughout the manuscript, but these changes were usually not explained or justified in the text (sometimes this information was found in the figure legends). For example, some imaging was performed at 6 hpi and some was performed at 24 hpi, and this was not clearly justified. We think that providing this kind of information in the text would enhance readability. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2 ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] Figure 1: · The intravital microscopy in Figs.1b/1c were hard for non-experts to interpret. It might help the reader if the two-photon microscopy images were retrieved from the extended data and paired with the IVM images as these are much easier to interpret. Also, why was a different CFU dose used in Fig. 1a compared to Fig. 1b/c? · 1F – is a heatmap the best way to present this data? Some readers are quite adept at interpreting heatmaps at a glance, but others could use some more exposition. · 1j-1l: Why were A549 cells used for plaque assays but U2OS cells for immunofluorescence microscopy? Please justify the use of different cell types. Figure 2: · 2I - Figure label says 12 hpi but figure legend says 24 hpi, · 2K – This method of data presentation was viewed as unhelpful, as the plots on the right had individual data points that obscured the lines that aid interpretation. This turned into a discussion of the use of these plots throughout the manuscript. We think that these side-by-side plots could be replaced by single violin plots that provide the reader with better information at a glance. Figure 5: ● Here, the authors use isg15+/+ usp18+/+, usp18C61A/C61A, and isg15-/- interchangeably with WT, USP C61A, and ISG15 KO. We think that harmonizing labels throughout would help the reader. Furthermore, the superscripts in the former terms are difficult to read in the PDF even with high magnification. ● Are specific analyses performed using methods that are consistent with answering the specific question? · Generally, this was viewed as acceptable throughout. ● Is there appropriate technical expertise in the collection and analysis of data presented? · Generally, this was viewed as acceptable throughout. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? ● Most noticeably in Figures 2 f-h, the authors compare means with bar plots. Given that in these panels they also provide the data points overlaid with the bars, we think they mean to draw attention to the distribution of the data as well as the differences between test groups. These plots would instead be more succinctly presented as violin plots. The bars, on the other hand, should not be used for mean comparison, as they do not convey the distribution of the data as well as the mean. As detailed by Chenxin Li in this GitHub entry, there are other, better options. We think that instead of two graphs per panel, a single violin plot or at minimum a box plot is a better representation of this data. ● There are other instances of these plots (like Fig 1E) with data overlaying the bars. We think this one could also become a violin plot to neatly display the comparison. For some other plots like in Fig 4E the bars are not legible under the data points. This panel would benefit from being just bars with SEM or CI bars. Perhaps if the authors wish to show each individual data point, they may consider supplementing this piece with FigShare or another figure data repository. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. ● OK 2. Quality: Completeness (1–3 scale) SCORE = 2 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. ● Title: "ISG15-modification of the Arp2/3 complex restricts pathogen spread" - this is confusing as the main message is that aberrant hyper-ISGylation of Arp2/3 results in actin tail alterations (shorter, thicker) and failed bacterial segregation post-division, which nevertheless increases spread due to the formation of bazookas. In this light, 'restriction' doesn't seem like the right description of the change. It is certainly true that the ISG15 KO cells display an enhanced bacterial spread phenotype, but we didn't think this was the main take-home message. ● Conclusions could have been better supported by making better use of the ActA Lm strain that could not stably recruit Arp3. How does this strain perform in vivo in WT or hyper-ISGylation mice, in terms of spread and invasion in the liver? Similarly, the mutant Arp3 construct that cannot be ISGylated could have been better leveraged to confirm and extend abstract-level conclusions. ● We discussed the following statement: "More importantly, our data indicate that ISGylation of mediators of actin-based motility acts a critical host defense strategy for both bacterial and viral pathogens, when properly regulated", This statement needs more support, as there are only Lm and VACV included in the paper, the VACV studies were underdeveloped, and the definition of "properly regulated ISGylation" was not well described. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. ● We did not identify a fatal flaw in the study, but we were most concerned about the fibroblast/epithelial layer studies presented in Figure 5. For example, while there were clearly changes in the fibroblast actin cytoskeleton, shape and motility that could be plausibly linked to ISGylation of the Arp2/3 complex and global defects in actin branching, only the motility phenotype was clearly linked to Arp2/3 complex, and this information was relegated to the extended data. 3. Quality: Reproducibility (1–3 scale) SCORE = 1.5 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? ● Fig2 – there was some discussion about whether more biological replicates for Figs2A-C were required to help understand the high variation in Arp3 localization on Lm in the hyper-ISGylation cells. There was some concern about the statistical tests per

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6807984. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Attenuation of a DNA Cruciform by a Conserved Regulator Directs T3SS-1 mediated virulence in Vibrio parahaemolyticus Landon J. Getz1, Justin M. Brown1, Lauren Sobot1, Alexandra Chow1, Jastina Mahendrarajah1, Nikhil A. Thomas1,2 doi: https://doi.org/10.1101/2022.03.07.483294 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Vibrio bacteria cause millions of life-threatening infections each year. These pathogens possess numerous disease-causing pathogenicity factors. Vibrio also encode DNA binding proteins that regulate virulence gene expression by altering DNA conformation. The HlyU family of winged helix-turn-helix (wHTH) DNA-binding proteins positively regulate a subset of virulence genes; however, the mechanism remains unknown. Several Vibrio strains have shown HlyU proteins outcompete binding of virulence genes by Histone-like Nucleotide-Structuring protein (H-NS) to prevent gene silencing. The HlyU and H-NS DNA binding sites do not often overlap, suggesting DNA topology or long-range protein-DNA interactions that could influence gene expression. The authors previously discovered that the exsBA intergenic region in Vibrio parahaemolyticus is an HlyU binding site that forms a cruciform DNA structure. DNA cruciform structures are present in all domains of life and have been implicated in repressing gene expression; however, functional DNA cruciforms in bacterial chromosomes are rare and no examples have been linked to virulence gene regulation. The authors aimed to investigate the mechanism of HlyU function in the context of exsA gene expression and subsequent T3SS-1 gene regulation. They hypothesized that a DNA cruciform structure is involved in regulation of exsA expression. The authors present data suggesting HlyU is responsible for attenuating a transcriptionally repressive DNA cruciform, leading to expression of exsA and resulting in V. parahaemolyticus T3SS-1 mediated virulence. They provide a stepwise model for the mechanism of DNA cruciform involvement in exsA activation and T3SS-1 activity. Overall, the authors present data suggesting a new role for HlyU in destabilizing a DNA cruciform structure that represses exsA expression, regulating virulence gene expression related to T3SS-1 activity. This reveals a previously overlooked de-repression mechanism of specialized DNA binding proteins to regulate gene expression. OVERALL ASSESSMENT: This is a well-written manuscript that provides new insights into the regulation of virulence gene expression for an important human pathogen. Conclusions are well supported by the data. There are significant elements of novelty throughout, increasing mechanistic understanding of roles for HlyU and DNA cruciform structures in virulence gene regulation. We identified opportunities for further increasing clarity to reach a broader audience. The authors should take this opportunity to discuss the significance and potential applications of this work. STRENGTHS: The writing is clear, and conclusions are well supported by the data. The authors provide new mechanistic insight into regulation of virulence gene expression. The novelty of this paper is a big strength as it suggests a previously unknown mechanism of gene regulation through a DNA cruciform structure and provides many new avenues for further research to understand virulence gene regulation in Vibrio spp. and other prokaryotes. WEAKNESSES: The only weakness we discussed was the supercoiling data, which if supported by further experiments could fit well within the dataset and provide stronger support for conclusions. In its current state, this data should be moved to the supplement, as it currently does not contribute to understanding of the model being established by the authors. Further explanation from the authors about the significance of the supercoiling data could help support its retention in the manuscript. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] o Figures 1A and 1B were hard to interpret without detailed reading of the figure legend. It would be easier to interpret if the corresponding plasmids and gels were paired in one panel along with adding the enzymes used in each lane. This would make scanning of the gel for the correct bands in each lane easier. This change in labelling could be applied to Figure 5A as well. o Figure 2 did not present results that strongly contributed to the storyline of the paper. By moving this figure to the supplementary data this would create a stronger, more linear story. Furthermore, supercoiling was not included in the model depicted in Figure 7, making it difficult for readers to understand the role it plays in this system. We suggest that sub-inhibitory levels of novobiocin may contribute to increased supercoiling; adding a reporter plasmid assay looking at linkage number changes would strengthen the supercoiling data. After the discussion in class and hearing from the authors about the role and significance of this figure to the story it was made clearer that this data is important. This should be clarified in the text so readers understand the full scope of this figure. o The scales for Figure 2C are not the same and the labels and legend are small relative to the rest of the figure. This could be improved to make reading easier. o In Figure 3C, it seems that there was a reduction on unbound DNA and slow shifting bands in IR2, IV1 and IV2 HlyU positive group. It is unclear what those bands are and why they were not considered as DNA-HlyU binding complex. o There was insufficient explanation about the S1 and S2 shift in Figure 3C. It wouldn't be much of an issue if both S1 and S2 disappeared in the mutant DNA group, but it may raise some questions in cases where only one band (S1 or S2) disappeared. ● Are specific analyses performed using methods that are consistent with answering the specific question? Yes ● Is there appropriate technical expertise in the collection and analysis of data presented? Yes ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? Yes ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. Yes 2. Quality: Completeness (1–3 scale) SCORE = ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o Generally good. o Including more specific key words in the title would better represent the findings in the paper. There was also some discussion about the use of the word "attenuation" in the title and if it was the best word choice. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o No. 3. Quality: Reproducibility (1–3 scale) SCORE = ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o Figure 6A and Figure 7C are missing statements in the figure legends that these experiments were repeated 3 times and a representative image is shown. This statement was appropriately included in all other figure legends. ● Is there sufficient raw data presented to assess rigor of the analysis? o Yes ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? o Yes ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o Yes ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o Yes ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant basis for decisions. o Non-expert readers would benefit from more in-depth groundwork in the Introduction. In general, while reading the Into, we found it difficult to conceive the antagonistic activity of HlyU and H-NS, how this controlled expression of exsA, and how this then related to T3SS activity. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o As the authors stated in their discussion, HlyU has shown de-repression of gene expression via competitive binding with H-NS. One of the novelties of this study is that it proposed another mode of action of HlyU: the binding of HlyU to its target DNA sequence has negative effect on the gene repression activity of DNA cruciform. This fundamental study provides a sufficiently important advance that does not require further extension. Impact: Extensibility (1–4 or N/A scale) SCORE = ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? o The relevance of Vibrio species was well established in the introduction; however, this is not touched on again in the discussion. The authors conducted their experiments using Vibrio parahaemolyticus, further experiments to recapitulate their findings identifying cruciform structures in other Vibrio species were included only in the supplemental material. It would have been nice to carry another species forward for further experiments, especially to further investigate how V. anguillarum performed since it was identified to have a cruciform structure in silico but did not linearize with T7 endonuclease. It would also be nice to have some interpretation from the authors as to the significance and application of their findings to combating diseases caused by Vibrio spp. ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6478660. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Stop codon recoding is widespread in diverse phage lineages and has the potential to regulate translation of late stage and lytic genes Adair L. Borges, Yue Clare Lou, Rohan Sachdeva, Basem Al-Shayeb, Alexander L. Jaffe, Shufei Lei, Joanne M. Santini, Jillian F. Banfield. 2021.08.26.457843; doi: https://doi.org/10.1101/2021.08.26.457843 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: The authors investigated expanded genetic codes in bacteriophage genomes. Bacteriophages from humans, pigs, baboons, cattle and horses were evaluated for TAG or TGA stop codon recoding. Alternatively coded genomes were more likely to contain TAG recoding than TGA and were found in low abundance in a human "western diet" cohort. To understand the phylogenetic relationship between families of alternatively coded phages, the authors constructed a tree of large terminase subunits from AC phages and standard code relatives. They found that TGA was often reassigned to tryptophan and TAG was often reassigned to glycine. Suppressor tRNAs allow for alternate coded organisms to access the translational machinery of hosts that use a standard genetic code. Almost half of the alternate code TGA- or TAG-using phages used suppressor tRNAs, suggesting this could be a mechanism used to overcome limitations of host translational machinery. Divergent stop-codon recoding strategies were observed amongst phage with very similar genomes suggesting rapid evolution of recoding potential. The frequency of alternative code usage in phage genomes supports a link between lysis regulation and code change. This link was termed the code change model for Jade phages and consists of a suppressor tRNA, a tRNA synthetase, and a release factor directly upstream of the lysis cassette. Overall, the investigation of alternative codon placement suggests that phages use alternate codes to control gene expression and timing. This is supported by placement of alternate stop codons in Garnet and Topaz genomes; these phages have alternate codons in lytic genes, as standard code does not provide stop codons for lytic genes such as integrase. This finding suggests that these phages alter the codon use of the host to regulate the latent/lytic switch. Overall, this pre-print provides evidence that stop-codon recoding in phages and prophages is used to expand the viral proteome. OVERALL ASSESSMENT: STRENGTHS: - The manuscript is well-written, and figures and data analysis are explained in the body of the paper, making the text easier to digest for the reader. - Overall conclusions are well supported by the data. - The dataset supports the hypothesis that phage use alternate genetic codes to control gene expression. WEAKNESSES: - The manuscript would benefit from additional information to explain the genetic code numbering system referenced in the paper (code 25 etc.) to non-expert readers. - We would like to see more discussion about what qualifies as a Western vs. Non-Western diet, and the limitations of the groups analyzed in this study. How well do they represent Western vs. Non-Western diets? - Differences in data presented from Western vs. Non-Western diets are initially discussed, but not mentioned in the paper's conclusion. To make the paper more cohesive, it would be beneficial to circle back to link new mechanistic understanding of phage stop codon recoding to the cohort studies. - Figure 2 is well done, but nevertheless it is challenging to interpret in its current form. We suggest providing a supplementary figure that explains the main findings from figure 2. - In Figure 1a, how do you deal with gene density when it is similar between standard and recoded phages? DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.5 ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] Figure 1. We think that additional information about the number of participants in each group should be included, and additional information about the diets they consume. We recognize that a Western diet is typically characterized by high fat intake, and the inverse for a Non-Western diet. We also recognize that the hunter-gather group has been previously published and is an accepted representative group for Non-Western diets that are commonly used in these types of larger bioinformatic studies comparing diet choice. However, more information on sample numbers, read depth and sequencing techniques would be quite valuable here. Figure 2. We think that the legend that accompanies the figure should be written in order of how data is presented on the figure. Right now, the legend does not follow the same organization as the figure it is describing. Many of the large phage clades are predicted to infect Firmicutes, which sometimes use alternative genetic codes. If alternatively coded phage infect hosts with alternative genetic codes, could it be argued that phages have adapted their codon usage to their hosts? If a host already uses an alternative genetic code, could this explain why some phages do not encode their own suppressor tRNAs? The results section for figure 2c has an extensive discussion about weaknesses/limitations of the dataset. This is important but overlong. Could some of this be moved to the Discussion, or paraphrased? Figure 3. We recommend providing an explanation regarding what the colors in 3b indicate (red vs. green sequences). We recommend indicating that partial genomes are shown in this figure. This information could be provided in the figure caption or results section. Figure 4. We recommend providing a statement in the results section that provides rationale about how representative data was selected and how many species from each clade were analyzed. These representative results are powerful, but we would like to see the generalized data as well so we can appreciate where these results were derived. Figure 6. The model is compelling but not yet supported by wet lab data. Could this be indicated somewhere (unless there is in fact wet lab data to support the model. ● Are specific analyses performed using methods that are consistent with answering the specific question? Yes, but we recommend a few extra supplementary figures to make data presentation clearer. ● Is there appropriate technical expertise in the collection and analysis of data presented? Yes. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? Yes, but we would like to see the generalized data from Figure 4. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. Yes. 2. Quality: Completeness (1–3 scale) SCORE = 1.8 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. The data supports the authors' conclusions. However, we suggest discussing alternative explanations for re-coding in phages. We do not yet know how efficiently amino acids are encoded by the AC codons and how often they are decoded as a stop codon. Perhaps the efficiency/frequency of recoding regulates abundance of target proteins. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. Unknown. 3. Quality: Reproducibility (1–3 scale) SCORE = 1.6 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?' N/A (Yes for Fig. 2). ● Is there sufficient raw data presented to assess rigor of the analysis?' Yes. ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? We suggest that more information be provided about database versions used and how many individuals were in the groups analyzed. Some methods sections do not describe the parameters used to run the programs. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? Authors could verify dietary analysis in secondary cohorts.' 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.2 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? We recommend exploring alternative rationales for AC usage, as described above. ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? We could not find another paper that examines stop codon reassignment in bacteriophages of gut microbiomes at this scale. The research presented in this manuscript is interesting and impactful, but authors could be more careful with their conclusion that stop codon re-coding can regulate lysogeny/lytic switch in phages until supportive wet lab data is obtained. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. See our comment regarding the legend in figure 2. MORE SUBJECTIVE CRITERIA (IMPACT) 1. Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.2 A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. This study provides key conceptual advances for the field, that could be extended by wet lab work to support Figure 6. 2. Impact: Extensibility (1–4 or N/A scale) SCORE = 2 ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? Yes ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). N/A

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6334820. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Defects in DNA double-strand break repair re-sensitise antibiotic-resistant Escherichia coli to multiple bactericidal antibiotics. Sarah A. Revitt-Mills, Elizabeth K. Wright, Madaline Vereker, Callum O'Flaherty, Fairley McPherson, Catherine Dawson, Antoine M. van Oijen, Andrew Robinson. bioRxiv 2022.01.24.477632; doi: https://doi.org/10.1101/2022.01.24.477632 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Building from previous literature, which demonstrated that inactivation of DNA repair pathways can re-sensitize bacteria to different antibiotics, the authors aimed to identify which DNA repair pathways are important for this re-sensitization and if this can be applied to additional classes of antibiotics. They utilized a collection of E. coli mutants that lacked important members of DNA repair pathways, including deletion of recAor recB genes involved in double-strand break repair (DSBR), or deletion of recF, recO, or recR genes involved in single-strand gap repair (SSGR), or deletion of mutT, which is important for the nucleotide pool sanitation (NPS) pathway. They then analyzed the resistance of these strains to various antibiotics using MIC test strips and spot plate assays. They found that disruption of the DSBR pathway enhanced the effects of ciprofloxacin and nitrofurantoin and re-sensitized resistant E. coli to these antibiotics. Further, they found that DSBR was important for bacterial survival during high concentrations of kanamycin treatment as they observed increased clearance of bacteria in E. coli strains lacking recA or recBcompared to wild type. This observation also repeated for kanamycin-resistant strains. In addition, deletion of recA, recB, or recO increased sensitivity to trimethoprim in both antibiotic-sensitive and antibiotic-resistant strains while deletion of recR only increased trimethoprim sensitivity in resistant strains. They next tested resistance to ampicillin and found no significant differences in sensitivity of DNA repair-deficient E. coli compared to wild-type parental strains. However, they observed a significant reduction in bacteria regrowth following ampicillin treatment in recA or recB-deficient cells, indicating that DSBR is important for bacteria tolerance to ampicillin. Furthermore, they examined induction of the SOS response in bacteria using an agar plate-based SOS reporter assay. They found that induction of the SOS response following ciprofloxacin or nitrofurantoin treatment required recB for both antibiotic-sensitive and -resistant strains, indicated by the decrease in fluorescence following antibiotic treatment. Lastly, the authors analyzed the sensitivity of DSBR-deficient E. coli to ML328 and IMP-1700, two DSBR inhibitors. They found that deletion of recAor recB increased sensitivity to these compounds and induction of the SOS response by these compounds is dependent on recA and recB. They also identified that IMP-1700 primarily targets topoisomerase IV while ML328 targets both topoisomerase IV and DNA gyrase, indicating that the DSBR pathway is not the only target of these compounds. In conclusion, the authors found that inhibition of DSBR can re-sensitize resistant E. colito various antibiotics. OVERALL ASSESSMENT: STRENGTHS: This preprint is very well written and provides ample relevant background to aid general-interest readers. We particularly appreciated how the literature was re-addressed in the Discussion in light of new findings, providing context for future planned studies and potential applications of these discoveries. WEAKNESSES: While there were many strengths in this pre-print, we also identified a few weaknesses we would like to address. One of our major concerns was the lack of discussion about the differences between wild-type E. coli and mutant strains. We wanted to know if there were differences between the growth rates of these strains in the absence of antibiotic challenge, to establish a baseline for these strains which could then be compared during antibiotic treatment. Additionally, we worried that changing assay formats from liquid cultures to solid plates between experiments might affect the findings. Indeed, changing how the bacteria are grown can introduce environmental differences which may affect growth rates, stress responses, and antibiotic effectiveness, all criteria which were measured in this pre-print. Some discussion of the strengths and weaknesses of the different assay formats is warranted. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2 ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] o For Figure 1, we questioned why there were a different number of replicates between conditions in panels A and D. We recommend having the same number of replicates shown per condition. o For Figure 2, we found the data to be inconsistent between panel B and panel C. We didn't understand why there was a significant increase in the ZOI from wild-type to the RecA or RecB mutant, shown in panel B, while there was no significant increase between these strains with vehicle-control in panel C. In addition, in reference to panel D, it was stated, "however at 10× MIC (Figure 2d) there was significant killing of both ΔrecA and ΔrecB mutants," yet no statistics were performed. While there is an obvious decrease in cfu/ml for the mutant strains, we think it is appropriate to use the word significant when statistics are used. We recommend either choosing another word or adding statistics to this graph to reinforce that the difference is significant. Lastly, we think the inclusion of the time-kill survival assay without antibiotic treatment would be a helpful control in panel D. o For Figure 3 and Figure 4, we had no critiques and thought they were well conducted and explained. o For Figure 5, we had concerns about the intensity of the fluorescent signal with the MIC test strips. The inferences provided were helpful to highlight the qualitative differences between the wild-type and mutant strains. However, quantification of this fluorescence intensity would be helpful to highlight quantitative differences between the activation of the SOS response in these strains. We also noticed a difference in the background intensity of the MIC strip itself in some of these images, specifically in panels C, D, and E. We questioned if this was a biproduct of the assay or if this was due to differences in camera settings or the acquisition of the images. Such differences could undermine the integrity of the assay. o For Figure 6, we wondered why there was a switch from use of the MIC test strips to use of the diffusion assays to assess SOS response activation with the DSBR inhibitors in panel G and H. An additional comment to explain the switch would be helpful for the reader. ● Are specific analyses performed using methods that are consistent with answering the specific question? o No, because there are changes in the methodology without explanation. ● Is there appropriate technical expertise in the collection and analysis of data presented? o Yes, however we recommend additional explanation of the methods used. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o Yes, except we recommend that the P-values from pairwise tests within the same experimental condition be controlled for family wise error rate. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o Yes. 2. Quality: Completeness (1–3 scale) SCORE = 2.5 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o No, because there is a disconnect between the data and the proposed major conclusions. The major conclusions stated in the title and the abstract suggest that targeting the DSBR repair pathway allows re-sensitization of these bacterial strains to various antibiotics. However, the data suggests that it is deletion of RecB specifically that confers this re-sensitization. We suggest that the major conclusions be adjusted to specify that RecB is responsible for this re-sensitization as opposed to the DSBR pathway in general. This is largely because the only other DSBR pathway component that was analyzed was RecA, and RecA is also involved in the SOS response pathway. If additional experiments analyzing other components of the DSBR pathway also showed re-sensitization to these antibiotics, then the proposed major conclusions would be justified. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o Yes, there are two experiments that if performed could disprove the conclusion. One experiment would be looking at other DSBR repair components, like RecC, RecD, among others, to see of their deletion would have similar effects as deletion of RecB. This would confirm that interference with the DSBR pathway is important for re-sensitization of these antibiotics as opposed to deletion of RecB specifically. Additionally, it would be important to see if the results would be consistent between growth in liquid culture and growth in solid media. This experiment would confirm that differences in antibiotic sensitivity are due to interference with the DSBR pathway and not differences in growth environments. 3. Quality: Reproducibility (1–3 scale) SCORE = 2.5 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?' o As mentioned above, the number of biological replicates performed for different experiments varied considerably throughout the figures for no clear reason. In addition, and as mentioned above, there appeared to be differences in the camara settings and picture quality between images taken for the SOS reporter assay. Consistent camera settings are required to truly make quantitative comparisons. ● Is there sufficient raw data presented to assess rigor of the analysis?' o Yes. ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? o Yes. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A. 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o Yes. ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o Yes. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. o One comment we have is about the color scheme used for the bar graphs. Some of the lighter shades used are very light and, at times, indistinguishable from the neighboring bar in the graph. This is a minor point but changing the color shade or including an outline around the bar would be easier for the reader to see the data. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 3 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.] ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o The idea that antibiotics induce DNA damage has been well documented in the field and there are multiple papers that address the DNA-damaging properties of the antibiotics mentioned in this pre-print. The novelty of this pre-print is in the idea of interfering with bacterial DNA repair mechanisms to render pathogenic bacteria susceptible to antibiotics, specifically by targeting RecB in the DSBR pathway. The proposed major conclusions of this preprint suggest that the defect of the DSBR pathway is the dominant cause of the antibiotic-sensitizing effects. If so, then the novelty of this preprint is lacking. Remodeling of the major conclusions to specify defects in RecB to be the major cause of the antibiotic-sensitizing effects, a target that has not been previously investigated to our knowledge, would greatly increase the novelty of this pre-print. 2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? o N/A ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). o N/A

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/6259304. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: The novel anti-CRISPR AcrIIA22 relieves DNA torsion in target plasmids and impairs SpyCas9 activity. Kevin J. Forsberg, Danica T. Schmidtke, Rachel Werther, Ruben V. Uribe, Deanna Hausman, Morten O. A. Sommer, Barry L. Stoddard, Brett K. Kaiser, Harmit S. Malik bioRxiv 2021.09.28.317578; doi: https://www.biorxiv.org/content/10.1101/2020.09.28.317578v2 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: A functional screen for SpyCas9 antagonism led to identification of AcrIIA22 as an anti-CRISPR protein. AcrIIA22 antagonizes SpyCas9 through a unique mechanism - instead of binding to the Cas protein, AcrIIA22 ORF1 functions as a nickase that cleaves supercoiled plasmids. This cleavage causes a release of the torsional stress held by a supercoiled plasmid, which could render these plasmids less susceptible to SpyCas9 targeting. In a trial using phage Mu, AcrIIA22 partially protected against SpyCas9-mediated restriction, in addition to completely protecting plasmids from SpyCas9 targeting and cleavage. AcrIIA22 has no known sequence homology to proteins with known functions, but AcrIIA22 homologs were identified in prophage genomes and small hypervariable regions in bacterial genomes. AcrIIA22 homologs were located near the end of prophage genomes, close to the junction with host genomes, providing insight into gene ancestry. There was evidence that AcrIIA22 promotes recombination in hypervariable bacterial "genomic islands" in the absence of other gene products that typically induce recombination. Most AcrIIA22 homologs were found in bacteria in the CAG-217 genus, and upon further analysis these homologs also inhibited plasmid targeting by Cas9 systems. Thorough analysis revealed that AcrIIA22 did not function via previously described anti-CRISPR mechanisms. The crystal structure of AcrIIA22 was solved, which revealed structural similarity to PC4-like proteins. AcrIIA22 oligomerization was necessary for protection against SpyCas9. Like PC4-like proteins, AcrIIA22 altered DNA topology through the observation of a change in a target plasmid's migrating form during gel electrophoresis from supercoiled to open-circle. Further in vitro analysis of purified AcrIIA22 protein revealed that AcrIIA22 nicks supercoiled plasmids to protect them from SpyCas9 activity. This function could be inhibited via the mutagenesis of key amino acid D14 to alanine, consistent with observations of a AcrIIA22 homolog with diminished nicking activity (AcrIIA22a). OVERALL ASSESSMENT: This pre-print (now published in PLOS Biology) describes a novel ACR mechanism and represents a significant advance in the field. Data was clearly presented in figures and text and the writing was engaging and carried the reader along. Authors used appropriate methodology and data analysis and overall data quality was viewed as a strength. Final conclusions about phage protection by this novel ACR protein could have been more strongly supported by studies of additional phage that feature a supercoiled genome topology that could be a good substrate for AcrIIA22. STRENGTHS: - Clearly presented data, easy to understand. - Strong writing, accessible to non-expert reader. - Identification of a novel ACR mechanism that is not so easily defeated by rapid microbial evolution. WEAKNESSES: - Conclusions about phage protection by this novel ACR protein would have been more strongly supported by studies of additional phage with supercoiled genomes. - The Discussion could benefit from some speculation about mechanisms of plasmid nicking. Is there a specific sequence that AcrIIA22 recognizes on the target DNA, or is the nicking carried out with no discrimination other than the target must be supercoiled? Does AcrIIA22 cause single-stranded breaks or are double-strand breaks possible? If so, what is the consequence for the phage/plasmid? DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 1 (by the way, 1 is high quality; 3 is low quality) ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] Figure 1: Data proving SpyCas9 expression is not affected by ORF_1 is important enough to move from the Supplement into Figure 1. Minor point: Graph axis labels need to be kept consistent. Figure 2: Good. We thought that the graphical representation of the data aided understanding of the conclusions. Figure 3: Good. We appreciated the data showing the ability of AcrIIA22 to inhibit other Cas9 systems and the location of the AcrIIA22 homologs in the CAG-217 genus. Figure 4: Good. We valued the visualization of the structure of AcrIIA22 and how that revealed the similarities to PC4-like proteins. Figure 5: Good. One reader had a minor comment about the differences between the 0.6 uM and 0.3 uM treatment groups, as there appears to be some variation in the starting plasmids compared to the other groups. Figure 6: Good. We noticed that in 6C there was still a slight shift of the plasmid into the OC state in the D14A mutant samples. We were curious if there was any speculation as to what may have caused this shift. An additional minor note is that some readers commented that the subtle greyscale colour scheme made the interpretation of the data more challenging. Figure 7: Good. We thought that the graphical data representation aided our understanding of the results. Additionally, we thought the data clearly outlined that SpyCas9 is more likely to target DNA that hasn't already been nicked by AcrIIA22. ● Are specific analyses performed using methods that are consistent with answering the specific question? We thought the experimental approaches were sound and were used appropriately to address research questions. ● Is there appropriate technical expertise in the collection and analysis of data presented? Yes, appropriate technical expertise was demonstrated. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? We had no issues with the statistics used throughout this paper. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. While this paper was an extension of a previous functional screen – we thought that there was sufficient literature cited throughout the paper, and there was enough support and evidence on the comparison between PC4-like proteins and AcrIIA22 function to justify the authors' conclusions. 2. Quality: Completeness (1–3 scale) SCORE = 1.5 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. We thought the data supported the abstract-level conclusions. It was clear that AcrIIA22 is able to relieve DNA torsion (changing topology from supercoiled to open circle) and this can impair SpyCas9 targeting against plasmids. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. While this research is complete, we thought that it would have been supported more strongly by determining whether AcrIIA22 could protect other phage that feature supercoiled viral DNA as part of their replication cycle. We also thought that the study would benefit from an exploration of the nicking mechanism. 3. Quality: Reproducibility (1–3 scale) SCORE = ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? Yes, there were sufficient replicates and experimental repeats. ● Is there sufficient raw data presented to assess rigor of the analysis? Yes, the supplemental information displayed good support to their figures. We especially appreciated the analytical gels and activity confirmation for the protein purification steps. Are methods for experimentation and analysis adequately outlined to permit reproducibility? Yes, there were no issues with how the methods were described. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? Use of the 2-plasmid kanamycin selection screen was used in a previous research project to identify AcrIIA22 as an antagonist for SpyCas9. This was further confirmed in this research through identification of ORF1 encoding the anti-CRISPR protein that functions through a unique mechanism to previously known ACRs. 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? Yes, the discussion was thorough and provided the proper context for the research. There was good speculation and discussion of alternative models that were not addressed in the dataset that could be used for future studies. ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? The citations were thorough and included many relevant research papers. We appreciated the use of citations for many of the minor points in the Introduction, as they allowed the reader to further investigate any area to gain a fuller understanding. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. We appreciated the well-written and concise nature of the introduction. We also thought that the Introduction would have benefited from additional details on specific examples of other ACR's and their mechanisms of action (are there other Acrs that have mechanisms besides Cas binding?). Additionally, adding in a biochemical section discussing protein structure and how it relates to suspected functions, like nicking, would have also aided understanding. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. The work presented in this paper was nicely put into the proper context of the field in terms of describing the known CRISPR-Cas evasion mechanisms and the novel understanding of Acrs and their functions. The screening approach and the methodology of determining the exact antagonism mechanism were easy to understand. AcrIIA22's evasion mechanism of relieving supercoiling topology was very well described. One of the most significant points that we discussed was that the research here shows that there are a wide variety of mechanisms of CRISPR immunity evasion that have yet to be discovered. ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. The research shows the strong inhibition of CRISPR-Cas targeting of supercoiled plasmids, and then the less potent protection of the phage Mu by AcrIIA22. While there is a well-reasoned argument for the advantages of this moderate CRISPR-Cas inhibition included in the Discussion, we thought that extending the analysis to other phage infection systems would strengthen this point. The point was made that the life cycles of native CAG-217 phages are not well understood at this time, and that there are many dsDNA phage genomes which undergo circular topologically restrained stages of their replication cycle, but the implication is that these native phages are better target mobile genetic elements for AcrIIA22 and including those in a protection assay may add considerable strength to the argument for the anti-CRISPR function of AcrIIA22. 2.Impact: Extensibility (1–4 or N/A scale) SCORE = ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). N/A, but we are very excited and interested to hear if there will be additional studies to gauge the effects of AcrIIA22 on defense against other phage infections – particularly if there was a test to examine a phage that has an important supercoiled form during some part of its lifecycle.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/5895108. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes. Sandra Schifferdecker, Vojtech Zila, Thorsten G. Muller, Volkan Sakin, Maria Anders-Osswein, Vibor Laketa, Hans-Georg Krausslich, Barbara Muller. bioRxiv 2021.09.14.460218; doi: https://www.biorxiv.org/content/10.1101/2021.09.14.460218v2 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: HIV capsid uncoating mechanisms remain poorly understood. Recent advanced microscopy studies have revealed intact HIV capsids in the cell nucleus, as well as transiting the nuclear pore complex (NPC). These studies revealed that the diameter of the NPC is greater than previously appreciated, and sufficiently wide to allow HIV capsid entry. While these advances in microscopy enabled remarkable visualization of these processes, studies to date remain limited by the ability to label and track HIV capsids, which are not very amenable to traditional methods of immunostaining or engineering fluorescent fusion proteins. Here, Barbara Muller's group reports on the expansion of the genetic code of HIV to allow site-specific incorporation of a non-canonical amino acid into the capsid (CA) protein via amber suppression. HIV particles were treated with a membrane-permeable fluorescent molecule that reacted with the non-canonical amino acid in CA, rendering the particles intrinsically fluorescent. This fluorescent HIV was used to track HIV entry into a HeLa cell-based reporter cell line, as well as primary patient-derived T cells. These fluorescent HIV virions were slightly delayed in trafficking to the nucleus compared to parental HIV. High MOI infections demonstrated that HIV particles accumulated in clusters in the nucleoplasm. These capsids were largely intact, based on measurements of fluorescence intensity of particles in the nucleus compared to the cytoplasm, and the known stoichiometry of capsid-associated CA compared to total CA in HIV particles. Cryo-EM studies of infected T cells revealed that these largely intact HIV capsids in the nucleus and nevertheless somewhat deformed compared to canonical bullet-shaped capsids. This preprint clearly establishes the feasibility of HIV capsid labelling via genetic code expansion and takes important first steps towards characterizing fluorescent capsids in the nucleus. This technology should enable future mechanistic studies of HIV uncoating. OVERALL ASSESSMENT: This preprint represents a strong technical advance in the HIV field, which will enable future studies of capsid trafficking and uncoating. It will be particularly valuable when using advanced microscopy methods. Incorporating methods to track HIV genomes in the context of these studies will further advance the application of this technology. The manuscript is very well written, and most messages are clearly conveyed, although the reader would benefit from a more thorough explanation of GCE and capsid labeling methodology. Studies were generally well supported by appropriate numbers of biological and technical replicates, and statistical tests. Overall data quality was good, although some data that had been relegated to the supplemental material could be retrieved to the main figure set to support the authors' conclusions more clearly. STRENGTHS: - Innovative approach and strong technical advance and starting place for future studies of HIV capsid trafficking and uncoating. - Quantitative evidence for intact capsids in the nucleus based on CA*(SiR) signal intensity in the nucleus compared to the cytoplasm was viewed as a very clever approach that supports recent microscopy data in field. WEAKNESSES: - Could be strengthened by a more fulsome explanation (with cartoons) of GCE and click-labelling methodology, and its application to HIV capsid labeling specifically. - DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 2 ● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.] o Figure 1A: The GCE and capsid labeling methodologies really deserve a standalone figure of their own, and more explanation in the body text so the reader can fully appreciate how cool it is. Otherwise, it goes by too quickly. In class, we also discussed whether the Vpr amber stop codon was the only one in the HIV genome that needed to be changed to set up the CA ORF mutagenesis and GCE. Please clarify. A genome map marking relevant features including Vpr ORF and CA ORF could be helpful. The current cartoon image in Figure 1A is low-resolution, which also makes interpretation more difficult. o Figure 1B: The representative TEM images could be improved by labelling features that the reader should notice. Images of budding viral particles are common in the HIV field, but general interest readers might not get everything out of the images that you want them to. o Figures 1C/1D/1E: We wondered why there was lower yield of the HIV-1*CA14-SiR particles in Figures 1C/1D, but comparable or higher levels of viral structural proteins in Figure 1E. Please clarify. Also, please label the ~48 kDa additional CA band in the HIV-1*CA14-SiR lane in Figure 1E. o Figure 2: Good. Minor point: some readers found the figure layout was a little difficult to follow at first and missed that Figure 2A is the quantitation for Figure 2A. Maybe consider revising layout for greater clarity. o Figure 3: Good. We wondered whether the particle quantitation in different cellular compartments in Figure 3D would be more informative in the form of violin plots as elsewhere in the manuscript. Were statistical tests performed on this data? o Figure 4: Good. We thought that the greyscale data in Figure 4C would be clearer if presented in colour. o Figure 5: There was considerable discussion about the use of the small molecule inhibitor PF74 as a control in Figure 5C. PF74 has been reported to interfere with capsid binding to host nucleoporins and the CPSF6 protein and reported effects range between capsid destabilization to having no effect on nuclear accumulation of capsids. Here, according to the figure legend, infected TZM-bl cells were treated with DMSO vehicle control or 15 uM PF74 for 1 h prior to fixation at 17 hpi. The result was that there was no significant effect on the nuclear accumulation of fluorescent capsids in the presence of PF74. However, the authors' statement about PF74 displacement of CPSF6 from capsids was unsupported by the data provided. The PF74 treatment was quite brief at only 1 h prior to fixation, and the reader was not given any rationale for this drug dosage or timing. Overall, the data in Fig 5C could be strengthened by using a range of drug concentrations and validating the displacement of CPSF6 that was inferred by the authors. o Furthermore, readers thought that the capsid cartoon in Figure 5A could be improved to provide a stronger link to the data in Figures 5B and 5C. The description of the excess CA proteins that were not incorporated into the capsid proper was surprising to some readers who were less familiar with HIV literature. Could elements of this cartoon be incorporated into a revised Figure 1 describing the GCE/click-labelling procedure in greater detail? o Figure 6: In Figure 6A, claims that CPSF6 dissociation is enabling binding of antibody should be better supported. CPSF6 immunostaining could strengthen this point. At least the authors should discuss this if there are technical reasons why this was not done. o Furthermore, one of our students who has some experience with Cryo-EM commented on the representative CLEM-ET data in Figure 6D-6E. They were hoping for more detailed descriptions of the methods. They also thought that whileisosurface rendering gave general structural information about capsids, subtomogram averaging could give more details about the capsid and may reveal whether they are truly largely intact, as indicated by the CA*(SiR) quantitation in Figure 5b. o Minor point: white text on ET images is difficult to see. ● Are specific analyses performed using methods that are consistent with answering the specific question? o The experimental approaches were generally sound and addressed the specific questions. o We thought that the experiments involving the PF74 drug needed better explanation and an approach that directly tested effects on CPSF6 binding, which was mentioned in the text. ● Is there appropriate technical expertise in the collection and analysis of data presented? o Appropriate technical expertise was demonstrated. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o Generally good throughout. Statistical comparisons were appropriate. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o The experimental foundations of this study are generally solid. The authors cited the literature appropriately and presented data that largely supported their conclusions. The technical achievements described in the manuscript affirm other newly emerged data in the field regarding intact HIV capsids in the nucleus. 2. Quality: Completeness (1–3 scale) SCORE = 1.5 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o Generally, the data supports the conclusions as stated in the title and abstract. However, we thought that the conclusions of the experiments involving the PF74 drug needed better supporting data. o We also wondered whether the delayed nuclear entry of click-labelled capsids might be a limitation of this technology that should have been explored and discussed further. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o N/A 3. Quality: Reproducibility (1–3 scale) SCORE = 2 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o Sufficient biological and technical replicates were performed to provide a high-confidence dataset. ● Is there sufficient raw data presented to assess rigor of the analysis? o This was a weakness in the manuscript. While representative images were clear and supported the main findings, some excellent supporting data was buried in the supplementary file. We believe that the manuscript would tell a more compelling story if some of the strong supplementary data were retrieved and presented with the main figures. o We also thought that there was insufficient raw data in the CLEM-ET experiments. Are methods for experimentation and analysis adequately outlined to permit reproducibility? o The methods are generally clearly described, but as mentioned above, more details and a detailed figure about the GCE and click labelling experiments would be very helpful for the general reader. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o The experiments in primary T cells serve as a validation of the HeLa cell studies. 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o Yes ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o Yes, but a more detailed coverage of the GCE/click-labelling as requested may necessitate more citations from the literature. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. o Overall, the paper was very well written and ideas were clearly conveyed, except in situations noted above, and repeated here: o Figure 1A: The GCE and capsid labeling methodologies really deserve a standalone figure of their own, and more explanation in the body text so the reader can fully appreciate how cool it is. o Figure 1B: The representative TEM images could be improved by labelling features that the reader should notice. o Figure 2: Some readers found the figure layout was a little difficult to follow at first and missed that Figure 2A is the quantitation for Figure 2A. Maybe consider revising layout for greater clarity. o Figure 3: We wondered whether the particle quantitation in different cellular compartments in Figure 3D would be more informative in the form of violin plots as elsewhere in the manuscript. o Figure 5A: Furthermore, readers thought that the capsid cartoon in Figure 5A could be improved to provide a stronger link to the data in Figures 5B and 5C. The description of the excess CA proteins that were not incorporated into the capsid proper was surprising to some readers who were less familiar with HIV literature. Could elements of this cartoon be incorporated into a revised Figure 1 describing the GCE/click-labelling procedure in greater detail? o Figure 6: Minor point: white text on ET images is difficult to see. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. o The key finding can definitely be understood by a layperson, although more helpful cartoon figures would make the genetic code expansion strategy more clear. ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o This manuscript represents a considerable technical advance that opens up new possibilities to investigate capsid trafficking and uncoating and takes some initial steps towards applying the technology. The technology was not fully extended and used to address such questions in this paper. 2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of valu

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768672. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Rachel Eguia, Katharine H. D. Crawford, Terry Stevens-Ayers, Laurel Kelnhofer-Millevolte, Alexander L. Greninger, Janet A. Englund, Michael J. Boeckh, Jesse D. Bloom. A human coronavirus evolves antigenically to escape antibody immunity. bioRxiv 2020.12.17.423313; doi:https://doi.org/10.1101/2020.12.17.423313 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Egula et al. investigated the ability of human sera collected over decades to neutralize contemporary and non-contemporary strains of HCoV-229E (229E). Using sera and viruses collected from the late 1980's to the present, the authors found that human sera from 1982 was unable to neutralize subsequent generations of 229E viruses, whereas sera recently collected from patients alive and likely exposed to 229E strains from 1982 could neutralize both modern and historic viruses, thereby providing some evidence for durable immunity. Finally, the authors confirmed that most of the mutations that prevent neutralization of modern viruses were found in the 3 loops of the receptor binding domain, although mutations in the N-terminal domain also likely contribute to antigenic escape. OVERALL ASSESSMENT: This is a good article with solid experiments and conclusions. We appreciate the way the article was written; it is clear and concise and follows a sound logical progression. However, we are not convinced that the work is particularly novel, although this may be an issue of framing (see final section on Subjective Criteria, below). STRENGTHS: The manuscript is well written, with a logical flow of ideas. Bioinformatics and neutralization data appear to be robust. Overall, while we have some concerns about statistics, the data is convincing. WEAKNESSES: The narrowing of sera samples for analysis seems to limit the scope of the study, and we think it might bias the results. Because of this, we are unsure if this data can be generalized to a larger population, or if it is only applicable to those whose antibody responses mirror the cutoffs chosen in this study. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 1.5 ● Figure by figure, do experiments, as performed, have the proper controls? - Fig. 1A: Since the authors have identified regions for each strain, they should justify relevance of those they selected compared to the region from which the sera they are testing are coming from. For instance, is it likely that the individual's sera tested would be exposed to viruses from Australia, China and the USA and if not, how would the phylogenetic divergence affect neutralization studies? It would have been helpful if the authors had also compared neutralization between closely related viruses from the same regions to determine if the sera was better at neutralizing certain spikes than others they selected. o Furthermore, for the figure legend, the distance bar labelled 4 years is not described in the figure legend and this would help the reader follow along. - Fig. 2C: The fold change representation of neutralization titers has a lot of variance and is difficult to determine any overall result from this figure. - Fig. 4B: The authors should have followed this experiment using NTD chimeras, as changes in the NTD of Spike may have clarified why, for some individuals, the RBD chimera had higher neutralization responses than the full Spike. o Furthermore, without stats on this figure how can the authors claim that "neutralization activity was rapidly eroded by antigenic evolution" considering that some of the sera did not see change. This is not a convincing claim without statistics. ● Are specific analyses performed using methods that are consistent with answering the specific question? - Fig. 2: There are no stats performed for neutralization titers. Is it common practice in the field to conduct only one replicate for these assays? Conclusions could be strengthened by studying multiple replicates (N=3) and applying appropriate statistical tests. ● Is there the appropriate technical expertise in the collection and analysis of data presented? - Yes. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? - Yes. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. - Yes. 2. Quality: Completeness (1–3 scale) SCORE = 1 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems, - Yes, the conclusions are well supported by three experimental systems: evolutionary analysis, human sera neutralization assays, and chimera spike receptor neutralization analysis. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. - There is no positive control for the neutralization assays. It would be beneficial to show the reader that these assays perform as expected against known antibody/antigen combinations. This would show the reader that the authors know what to expect from their other neutralization assays. 3. Quality: Reproducibility (1–3 scale) SCORE = 2 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? - Repeats were not performed for the neutralization assays. ● Is there sufficient raw data presented to assess rigor of the analysis? - Yes. ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? - Yes. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? - N/A. 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? - Is it possible that the decrease seen in the older patients for the neutralization of historic viruses could be related to immune senescence or some immune amnesia-like phenotype from a different infection? This might be worth exploring in the discussion as it is an interesting data point. ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? - Yes, the authors did a wonderful job explaining that there is little work in this area and highlighting why they believe their work is novel. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. - Fig. 2: box plot could perhaps be formatted differently. As it is shown, it seems to work against the authors conclusions. Also, were stats performed on this box plot? If they are, they should be shown. Stats would make the plot more understandable in context of your other data. MORE SUBJECTIVE CRITERIA (IMPACT) 1. Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. - This article is interesting and provoked a great deal of discussion in our journal club. It advances our current understanding of antigen evasion by viruses (specifically coronaviruses) and our immune system's ability to detect and neutralize historic coronaviruses. It also confirms that coronaviruses mutate largely in the receptor binding domains, but not as much elsewhere, to evade the immune system. - However, we have not awarded full points on novelty because on some level we think that the results are to be expected. People who have been exposed to a virus likely have neutralizing antibodies against them. People who haven't likely do not. Because this takes up most of Figures 2 and 3, we believe this is a major conclusion of the paper. - With that said, this remains foundational work for understanding human serum neutralization capacity for coronaviruses over time. A greater emphasis on the implications for durable anti-coronavirus immunity in the Discussion and elsewhere may strengthen the perception of novelty. ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o N/A 2. Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). o N/A

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768659. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Human coronaviruses disassemble processing bodies. Carolyn-Ann Robinson, Mariel Kleer, Rory P. Mulloy, Elizabeth L. Castle, Bre Q. Boudreau and Jennifer A. Corcoran. bioRxiv 2020.11.08.372995; doi: https://doi.org/10.1101/2020.11.08.372995 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Robinson, et al., investigated the effects of human coronavirus OC43 infection on RNA granules known as processing bodies (PBs). They demonstrated that HCoV-OC43 infection reduces numbers of visible PBs in human umbilical vein endothelial cells (HUVECs). They observed that overexpression of the PB-resident mRNA-decapping enzyme Dcp1a prior to HCoV-OC43 infection restricts viral replication in HUVECs through an unknown mechanism. Using plasmid vectors expressing individual SARS-CoV-2 genes, the authors identified several candidate genes that caused PB loss. They screened the same collection of viral genes for stabilization of a labile AU-rich element (ARE)-containing luciferase reporter, which often correlates with PB loss. They identified a non-overlapping set of hits in these two screens. Overall, this manuscript reports on a novel discovery that HCoV-OC43 infection induces PBs disassembly, possibly through the integrated effects of several virus-encoded proteins. OVERALL ASSESSMENT: STRENGTHS: The authors provided evidence that HCoV-OC43 infection correlates with loss of visible PBs. This is the first report of PB modulation by a HCoV. The identification of candidate SARS-CoV-2 genes that modulate PBs provides a useful starting point for future mechanistic investigations. The authors discussed the findings and the limitation of their study in detail and with appropriate support from the literature. WEAKNESSES: The manuscript could be improved by providing stronger rationale for selection of hits and each set of experiments as the reader moves through the Results section. There was concern that the antiviral effects of DCP1a overexpression could be due to DCP1a itself rather than a PB-specific effect. Further experiments are required to fully support the proposed model (see below). There was concern that while the data showed certain correlations, causality was not properly established in support of the model. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 2 · Figure by figure, do experiments, as performed, have the proper controls? (While discussing proper controls of the experiments, we also use this opportunity to discuss the rationale and approaches of each experiment) Figure 1: · Fig. 1B: Bars appear to represent the mean rather than SEM as stated in the Figure Legend. Please clarify. · Fig 1C: Inclusion of a loading control like actin would strengthen these western blots and account for any changes in protein expression due to protein loading differences. The total protein stain that was mentioned in the Materials and Methods could also be used effectively here. · In Fig 1A, ectopic expression of E protein appeared to result in more PBs, while it was showed as nonsignificant change compared to control in Fig 2B. Is the image in Fig. 1A representative? We think adding the SEM on Fig 1B and some author commentary on the proteins that led to increased PBs (especially E and nsp13) would be helpful to address the stringency of the assay. · In the Results, the authors mentioned that there were 6 SARS-CoV-2 proteins that mediate PB disassembly. However, only two of these met the standard of statistical significance in Fig 1B (N in thresholded group and nsp11 in unthresholded group). We are wondering what criteria were used to determine whether a protein was a significant hit or not in Fig 1B. The authors should describe these criteria clearly in the Results section. Figure 2: · Overall, these ARE-mRNA reporter assays are well described and appear to be properly controlled. Minor points: o In the Results section, the statement 'Of these ARE-mRNA regulating SARS-CoV-2 gene products' seems to be a strong conclusion just based on the luciferase experiment without confirming whether those proteins could regulate endogenous ARE-mRNAs. Is it possible for the authors to directly test the steady-state levels and stability of some endogenous ARE-mRNAs? o In the case of nsp1, is it fair to claim that the ARE-mRNA luciferase assay screen confirms the reduced levels of visible PBs in Fig 1B? This statement could be much better supported by data on endogenous ARE-mRNAs. o We noticed that the authors are currently validating some of the SARS-CoV-2 proteins in endothelial cells, which will provide additional opportunities to measure the accumulation and turnover rate of endogenous ARE-mRNAs. Therefore, we recommend that the authors add those data once available to strengthen their conclusion or simply tone down their conclusions based on the data included in the current pre-print. Figure 3: · No issues with this dataset. Figures 1-3: · There was considerable discussion and confusion about the rationale for moving forward with further investigation of N and nsp14 hits in HUVECs, rather than other candidate genes. The authors should make this rationale very clear, to avoid such confusion. o Based on the results of the screen, it seems the most prominent hit would be the ORF7b which was confirmed by both IF (Fig 1B) and by luciferase assay (Fig 2B), however it was not further investigated in HUVECs. N protein was shown to mediate PB disassembly by IF and was the top hit in the thresholded group but showed no elevation in the ARE-containing FLuc reporter. On the other hand, nsp14 was not one of the 6 hits from Fig 1B but showed significant elevation in the ARE-containing FLuc reporter, and it was not until the later validation in HUVECs showed nsp14 led to PB disassembly. In any case, further justification of these choices would be welcome, including how the results of two assays (IF and luciferase) were weighted in the decision. We also think the authors should provide some description of the known biological functions of these hits in the Results if this information was relevant to their decisions. We realize that some of this relevant information is in the Discussion, but it would be much more helpful to the readers to understand the data if some of this could be found in the Results section. Figure 4: · No major issues with experiment setup or controls. · One minor point is that in Fig 4C, the error bars for cytokine transcript levels are very large. It appears that there is only one replicate (especially for IL-8) that is pulling the average up. We realize this could be a genuine result. However, this data could be strengthened by inclusion of a few more biological replicates. Units should be shown on the y-axis label. Figure 5: · The purpose of overexpression of GFP-Dcp1a was to induce PB formation before viral infection. Could the authors quantify this effect to show that there is higher number of PBs in the GFP-Dcp1a expressing cells compared to the control GFP-expressing cells? · For Fig 5C, the lentivirus inoculum should be described in terms of MOI, so the reader understands how many infectious lentiviruses are being delivered to target cells at the 1x and 5x doses. There was also some discussion of the potential negative effects of higher MOI lentivirus transduction on coronavirus replication. To mitigate this concern, the authors could include an additional control with a high MOI infection with the GFP lentivirus. Also, the y-axis label is confusing. · Are specific analyses performed using methods that are consistent with answering the specific question? o Yes · Is there the appropriate technical expertise in the collection and analysis of data presented? o Yes · Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o Yes o One factor that may affect the results of the statistical analysis is the inherent false discovery rate associated with making so many comparisons in these screens. Careful attention to these statistical tests is warranted. · Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o The experimental foundations of this study are solid. 2. Quality: Completeness (1–3 scale) SCORE = 2 · Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o The authors made a novel discovery of human CoV-mediated PB disassembly, which is well supported by their results. However, more evidence is required to support the statement that stimulating PB formation prior to OC43 infection restricts viral replication. Specifically, the correlation between the overexpression of Dcp1a and PB formation were not clearly demonstrated in the study. In addition, these experiments to not make a distinction between a potential antiviral effect of Dcp1a vs. a potential antiviral effect of PBs. We suggest the authors may want to consider overexpressing other PB proteins such as DDX6, along with complementary approaches like silencing expression of Xrn1 to help assess whether PBs are antiviral. Moreover, the statement that 'disassembly of PBs enhances translation of proinflammatory cytokine mRNAs' in the abstract was not fully supported by the data in the study. Perhaps, the authors could consider using cytokine ELISA to demonstrate the enhance translation of cytokines such as IL-6 and IL-8. Alternatively, this statement could be removed from the abstract. · Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o No 3. Quality: Reproducibility (1–3 scale) SCORE = 1.5 · Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o There are inconsistency regarding to the N numbers in few figures: § Fig 3B and Fig 5B display inconsistent N number between treatment groups. Several experiments are only supported by 2 biological replicates. · Is there sufficient raw data presented to assess rigor of the analysis? o We think that providing the representative IF images for the strep-tag staining of each viral protein and the Raw data of the luciferase assay in supplementary would be very helpful for the audience to understand the rationale behind Figs 1-3. · Are methods for experimentation and analysis adequately outlined to permit reproducibility? o Yes, with one exception. The duration and the choice of antibiotic for selection after lentivirus transduction for GFP-Dcp1a overexpression in HUVECs was not clearly stated in the legend for Figure 5. · If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 2 · Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o The authors provided a very informative discussion including the limitations of the current study, speculations based on their data and published work, as well as their ongoing research work on further investigating the findings of the current preprint. · Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o The authors paid close attention to the literature throughout. · Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant bases for decisions. o There is room for some improvements in grammar and writing to enhance readability. o The authors may consider adding description of hits and rationale for selecting certain hits for further study to the corresponding results section. We believe this will help the readers to better understand their choices. o The authors mentioned 'Strep tag shown in red' in the legend of Fig 3A but it is actually green. MORE SUBJECTIVE CRITERIA (IMPACT) Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2.5 · A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. o The authors clearly made a novel discovery of HCoV-mediated PB disassembly, which contributes to our understanding of the human CoV-host interaction. · How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o The authors have included a model that proposes that HCoV replication is restricted by the PBs while the virus combats the PBs by inducing PB disassembly, which results in inflammatory cytokine production. If fully supported by the data, this model would be important as it revealed a novel role of PBs in host antiviral response in human CoV infection. However, based on the data provided, we think more work need to be done to support the proposed model. The evidence showed in the current study is not sufficient to demonstrate whether there is a direct link between PBs formation and the antiviral effects as well as the link between viral-induced PBs disassembly and the proinflammatory cytokine level. o The identification of candidate SARS-CoV-2 genes for virus-induced PB disassembly is useful as it could lead to the identification of some mechanisms SARS-CoV-2 manipulating the host upon infection. However, the incomplete characterization of these genes could be problematic. We believe by having one PB-disrupting CoV protein fully characterized for both PB and ARE-mRNA turnover modulation would greatly strengthen the conclusion. Impact: Extensibility (1–4 or N/A scale) SCORE = N/A · Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? &

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768668. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Poly(rC)-binding protein 1 limits hepatitis C virus assembly and egress. Sophie E. Cousineau, Selena M. Sagan. bioRxiv 2021.02.28.433252; doi: https://doi.org/10.1101/2021.02.28.433252 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Cousineau and Sagan explored the role of poly(rC)-binding protein 1 (PCBP1) in the HCV life cycle. Using the HCVcc system in Huh7.5 cells, they found that PCBP1 silencing decreased viral protein and viral RNA accumulation but increased virion production. After dissecting the viral replication cycle with HCVpp, a replication-defective reporter RNA, and assembly-deficient reporter RNAs, the authors concluded that PCBP1 silencing had no impact on HCV entry, translation, and genome replication. By contrast, inhibition of viral RNA synthesis with 2'CMA increased virion production from PCBP1 silenced cells, suggesting that PCBP1 may modulate HCV assembly and/or egress. OVERALL ASSESSMENT: STRENGTHS: The manuscript is concise, well-structured and straightforward. The finding that PCBP1 hinders a late stage in HCV replication is novel. WEAKNESSES: The precise mechanism of PCBP1 control of HCV assembly/egress was not fully explored in this manuscript. Data interpretation for Fig. 1, as well as the Methods for Figure 3 and Figure 5 may be confusing and require further exposition/clarification (described in detail below). The reader would benefit from additional context and discussion of our current understanding of HCV assembly/egress, as well as the known roles for PCBP1 in the cell. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 2 ● Figure by figure, do experiments, as performed, have the proper controls? o Fig.1: We suggest to include more details in the figure legend, such as indicating whether the HCV/actin quantification was normalized to siRNA control (Fig.1D) and spelling out "focus-forming unit (FFU)" (Fig.1E), to make the figure more self-explanatory. o Fig.2: It is not clear whether the authors used Renilla as an internal control here. We suggest that the authors clarify and present normalized data or include the control data when an internal control was used. o Fig.3: In the figure legend, authors stated that cells were electroporated with both RLuc and FLuc RNAs, therefore, fold change could have been calculated. We suggest the authors to include normalized data or explain why the control was not shown. The control may provide an indication of electroporation efficiency between samples. o Fig.4: Yes o Fig.5: Fig.5D shows an increased intracellular virus accumulation rate in PCBP1 silenced cells, which seems contradictory to the decreased viral proteins and RNA accumulation and unaltered intracellular titers in Fig.1. It would be nice if the authors discussed this discrepancy. In addition, we wondered if the results in Fig.5D would be different if the samples were collected beyond 12 h post-treatment. ● Are specific analyses performed using methods that are consistent with answering the specific question? o Fig.1: We wonder if the viral protein levels were increased in the supernatant? This would further support the hypothesis that decrease of intracellular viral protein accumulation was due to an increased viral secretion. o Fig.2: HCVpp expressing the glycoproteins of JFH-1T might be a better model for the purpose, considering that (1) JFH-1 and the H77 used here belong to different genotypes, and that (2) JFH-1T has adaptive mutations in the E2 coding region. o Fig.4: Quantification of viral RNA using qRT-PCR may be more accurate, and a comparison of replication kinetics between the packaging-defective viruses and the full-length virus could better demonstrate PCBP1's impact on packaging. o Fig.5: Blocking HCV egress using endosomal inhibitors such as U18666A or Bafilomycin-A1 (https://jvi.asm.org/content/84/21/11590) in PCBP1 silenced cells may directly show the impact of PCBP1 on HCV release. ● Is there the appropriate technical expertise in the collection and analysis of data presented? o Yes ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o Fig.5C and E: Statistical analyses were not performed. o Fig.5D and F: We suggest the authors to show the raw data in scatterplots and their regression lines, and specify how the accumulation rates were calculated. It would make the data much clearer. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o Yes 2. Quality: Completeness (1–3 scale) SCORE = 1.5 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o In general, the results support the proposed title and abstract. However, how PCBP1 affects HCV assembly or egress is unclear. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o Given the implications of PCBP1 on the turnover of MAVS, we wonder if these results could be reproduced in a cell line with RIG-I intact, such as Huh7 cells or primary human hepatocytes. 3. Quality: Reproducibility (1–3 scale) SCORE = 1.5 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o Yes ● Is there sufficient raw data presented to assess rigor of the analysis? o Fig.2 and 3: We suggest that the authors show the raw data and luciferase controls, or explain why they didn't include it for normalization. o Fig.5D and 5F: We suggest the authors show raw data in scatterplot as mentioned above. ● Are methods for experimentation and analysis adequately outlined to permit reproducibility? o For the transfection control in "HCV and VSV pseudoparticles (HCVpp and VSVpp)" and "Luciferase assays", the authors mentioned that Dual Reporter Luciferase kit was used, but all data were shown in RLU. We suggest authors clarify this and present normalized data when an internal control was used. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 2 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o Yes ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o We feel that more information on HCV assembly/egress and how PCBP1 could affect these processes would be quite helpful for the reader, both in the Introduction and Discussion. o While PCBP1's role in viral replication cycle was mentioned, more introduction on PCBP1's physiological functions / cellular role in hepatocytes and the poly(rC) targets of PCBP1 will also be helpful. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. o Line 62-line 63, the statement of 'a number of cellular proteins and RNAs have been shown to interact with the HCV genome.....' missing citation. o Bar graphs: Perhaps these results could be shown as boxplots. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 3 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. o This study examined the impact of PCBP1 on each step of HCV viral life cycle and proposed a potential role of PCBP1 in modulating HCV packaging and egress. In-depth analyses on how PCBP1 restricts HCV assembly will add to the impact of this study. ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. 2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). o We suggest this study could be extended to another cell line, such as Huh7, or primary human hepatocytes.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768664. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Exploring the interaction network of a synthetic gut bacterial community. Anna S. Weiss, Anna G. Burrichter, Abilash Chakravarthy Durai Raj, Alexandra von Strempel, Chen Meng, Karin Kleigrewe, Philipp C. Münch, Luis Rössler, Claudia Huber, Wolfgang Eisenreich, Lara M. Jochum, Stephanie Göing, Kirsten Jung, Alvaro Sanchez, Bärbel Stecher. bioRxiv 2021.02.25.432904; doi: https://doi.org/10.1101/2021.02.25.432904 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: Weiss et al., examine the growth rates, metabolic usage, and interaction patterns of the Oligo-Mouse-Microbiota synthetic community in an in vitro setting. They use this bottom-up approach to investigate the different metabolites used by various strains in the community, and the overlap of these metabolites between strains. They also show that some microbes like Enterococcus faecalis have strong impacts on the growth rates of other members within the community. Examination of E. faecalis specifically goes on to show that it could be due to cross-feeding events as well as the production of bacteriocins. Overall, the work shows that metabolite usage profiles along with the production of antimicrobials by community members can have a large impact on the observed community abundances in an in vitrosetting. OVERALL ASSESSMENT: STRENGTHS: -The authors provide several interesting interactions in their in vitro study that can be further investigated in vivo. -The authors do a good job at explaining the methods used in the paper. -The authors investigation into E. faecalis specifically was interesting and might give some information on how this microbe blooms after antibiotic use. WEAKNESSES: -Metabolic models were not validated. -While many of these findings are interesting it is unclear how well they will translate to the complex environment of the murine gut. -It would have been helpful to relate in vitro results to observations in vivo. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 2 · Figure by figure, do experiments, as performed, have the proper controls? [Note: in this section, the class discusses proper controls, but also uses the 'figure-by-figure' opportunity to discuss rationale and approaches] · Figure S1: Is it possible to look at these growth curves in different media types? A. muciniphila is dependent on mucin in the gut and may have different growth dynamics in media that contains mucin. Overall, we think that by examining multiple media types (or at least the addition of mucin) would improve the ability to translate these in vitro results to an in vivo setting. If this is not possible the authors should at least discuss the implications of using different media types and the lack of mucin in the media. · Figure S5: We found this figure difficult to read given the number of different shapes and colors displayed. Despite this, we believe that it conveys important information. Would it be possible to create a multiple plot panel showing the profiles of each strain separately? This would help with readability and interpretation of the figure. It may also be possible to highlight the area that shows key differences in metabolic activity for the reader to interpret. Finally, depending on the journal, would it be possible to include an interactive version of the plot? (Using an R library such as plotly) · Figure 1: Is there any phylogenetic relationship between how pH changes in DSM? For example, do organisms show similar pH changes when grown in SM from taxa in the same phyla, but then show different pH changes when grown in SM from different phyla? Is it possible to show whether these pH changes correspond with the overlap if metabolic usage between strains? o While not required it would be interesting to see whether the use of multiple bacteria in the lawn during the spot assay show the expression of the lanthibiotic from B. coccoides YL58. Alternatively, the B. coccoidesYL58 co-culture or its supernatant could be used for the spot assay instead of spotting the monoculture of YL58. o Minor point: it would be interesting to see if using SM instead of bacterial culture in the spot assay would inhibit bacterial growth. o These experiments rely heavily on spent media, but the gut is a somewhat nutrient-rich environment. While the spent media pairs nicely with the metabolomic work, we believe that the authors should mention this difference within the Discussion. o Color consistency between figures is really appreciated and helpful. We were wondering if more easily discernible colors should be used to represent the bacterial strains (the greens are quite similar to one another). o We believe the manuscript could benefit from the use of a colorblind friendly palette. · Figure 2: Are there metabolites depleted by all bacterial strains tested? If so we think this would be interesting to highlight. o Is the conclusion that A. muciniphila using a lower number of metabolites due to it using metabolites that are "novel" and not in the databases used to analyze the mass spec data? Its not clear whether annotated compounds were used in this analysis or all features with a mass:charge ratio and retention time were used. Clearing this up should address the above question. · Figure 3: We were confused about the description of the "draft metabolic model" in the manuscript. The authors should clarify whether this is a purely computational model. · Figure 4: We were wondering if the relative abundance of each strain in any pair at time 0 would be at least close to 50% (the blue and red bar) because the authors started with same OD of two strains and did 1:1 ratio mixing. We realize this could be due to many reasons such as different DNA extraction efficiencies, 16S rRNA copy number and different primer efficacies for different taxa. However, the authors should comment and clarify this result as we would expect some of them to be closer to 50:50 than shown in the figure. o The abundance of these microbes is referred to as "absolute abundance", which is clearly not true given the differences at time 0. Perhaps they should replace this with 16S rRNA gene copy number of 16S copy number throughout the text. · Figure 5: (Fig. 5B) Authors mention that strains YL2 and I49 did not grow in the co-culture, but provide no possible explanation as to why these strains did not grow. (Figs. 5B and 5C): strains YL44 and KB18 seem to be absent from co-cultures in both B and C, but this is not mentioned. Also, is KB18 present in the original inoculum? The authors mention they co-cultured all 12 strains, but the colored abundance representation for the "inoculum" does not include KB18. o While not necessary, it would have been interesting to see whether the compounds that contain C14 malate are those that the metabolic models from Figure 3) would have predicted. o The authors mention that compositional analysis was used in line 316 although the meaning of this statement is unclear. Furthermore, the authors are examining relative abundance data and therefore should not use terms such as "increased/decreased abundance". Instead, terms that refer to relative changes should be used instead. o We think Figure 5F may be missing a negative control for C14 (a different sugar that is not taken up by bacteria). Are specific analyses performed using methods that are consistent with answering the specific question? · The analyses performed in this study directly address the research question. Is there the appropriate technical expertise in the collection and analysis of data presented? · Yes Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? · Is a t-test appropriate for the analysis on metabolomic data? Perhaps the authors should use a non-distribution based tested such as a Wilcoxon ranked sum. Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. · For the most part, the authors do make use of controls when possible, however, the analysis of C14 malate uptake would be strengthened by comparison to a negative control. 2. Quality: Completeness (1–3 scale) SCORE = 2 Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. · Yes, the results do match the title and abstract. However, considering that this is an in vitro study, the title should be revised to convey this information more clearly. Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. · N/A 3. Quality: Reproducibility (1–3 scale) SCORE = 2 Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? · Information on the number of replicates for each experiment is scattered throughout the manuscript. Sometimes it could be found in a figure legend, whereas sometimes it can be found in the main text of the manuscript. This information should be clearly and consistently described in one section. Is there sufficient raw data presented to assess rigor of the analysis? · Yes, although the authors do not make any mention of where the metabolomic data or modelling data is posted for public use. Are methods for experimentation and analysis adequately outlined to permit reproducibility? · Yes – the metabolomics section is exceptionally thorough. If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? · N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 3 Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? · The authors do a reasonable job of addressing limitations of the study in the penultimate paragraph in the Discussion. However, we think that the manuscript would be strengthen by inclusion of an in-depth discussion of the impact of the selected media on the system, and to provide context on on future in vivo studies. Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? § Yes. Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. § More detailed information on the OMM would be helpful (how was it made?) § Lines 130-132 suggest that Figure 1D plots the magnitude of the differences in pH, but both the figure legend and the legend indicate that raw pH is plotted. MORE SUBJECTIVE CRITERIA (IMPACT) Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2 A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. § This manuscript will be of great interest to microbiome systems researchers. § The work is important because it demonstrates that complex in vitro microbiome systems can be constructed and intensively studied and yield hypotheses to be tested in vivo. § The authors grouped the 12 strains into three different growth rates and showed the depletion level of each metabolomic features in Figure 2A and the relative abundance in Figure 4, which likely correlate with the growth rate, but authors did not discuss the possible effects of the growth difference between strains on their interactions. The Discussion was more focused on the data that was less correlated to the growth rate such as the number of depleted metabolomic features of each strain and the absolute abundance of each strain in co-culture relative to monoculture. How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. · We discussed the impact of the paper and thought that it would be appropriate if the authors discussed whether the in vitro findings could be recapitulated in vivo and how much their studies can actually tell us about the OMM (considering that everything was done in vitro, and they cannot replicate the mouse gut environment in vitro). Impact: Extensibility (1–4 or N/A scale) SCORE = N/A Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? · N/A This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). · While not needed, we believe that the manuscript could benefit from the investigation of the OMM's ability to inhibit growth of enteric pathogens. We believe this experiment could be straightforward with the addition of an enteric pathogen to their cultured complex community.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768679. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations. Shane Miersch, Zhijie Li, Reza Saberianfar, Mart Ustav, James Brett Case, Levi Blazer, Chao Chen, Wei Ye, Alevtina Pavlenco, Maryna Gorelik, Julia Garcia Perez, Suryasree Subramania, Serena Singh, Lynda Ploder, Safder Ganaie, Rita E. Chen, Daisy W. Leung, Pier Paolo Pandolfi, Giuseppe Novelli, Giulia Matusali, Francesca Colavita, Maria R. Capobianchi, Suresh Jain, J.B. Gupta, Gaya K. Amarasinghe, Michael S. Diamond, James Rini, Sachdev S. Sidhu. bioRxiv 2020.10.31.362848; doi: https://doi.org/10.1101/2020.10.31.362848 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: In this pre-print, James Rini and Sachdev Sidhu's team applied a modular strategy to develop synthetic tetravalent antibodies against SARS-CoV2. Iterative changes improved the antibody specificity and binding affinity while retaining advantageous biophysical properties for drug development. Furthermore, the authors demonstrated that the antibody was effective against potential escape mutants. Thus, the authors provided a workflow for synthetic antibodies that can be readily applied against COVID-19 and other emerging viral threats. OVERALL ASSESSMENT: STRENGTHS: Overall, we think that the authors have designed a potent antibody candidate using the processes described in this manuscript. The manuscript is written concisely and describes design principles that can guide the engineering of synthetic antibodies with strong neutralizing capabilities against SARS-CoV2. The authors identified residues that confer improved antibody binding affinities and elucidated mechanism of action using electron microscopy and X-ray crystallography. Overall, a framework for rapid therapeutic antibody development was well described in this manuscript. WEAKNESSES: In more than one instance, we thought that analyses could be strengthened by the inclusion of a control antibody. The authors may consider an examination of the potency and biophysical properties of antibodies with specificity against SARS-COV2 (references are made to Bamlanivimab, Casirivimab, and Imdevimab in the introduction). The reader would benefit from a more detailed description of the construction of IgG1 antibodies 15031 – 15033 and the variants of IgG 15033 (data is only provided for 15033 and variants). DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 1.5 ● Figure by figure, do experiments, as performed, have the proper controls? ● [Note: in this section, the class discusses proper controls, but also uses the 'figure-by-figure' opportunity to discuss rationale and approaches] o Figure 1: We discussed at length how these experiments would be stronger by including an existing anti-SARS-CoV2 as a control, to provide a direct head-to-head comparison. Without controls, it was more challenging to assess performance of synthetic antibodies. ▪ Would Figure 1B be better placed as the last panel for clarity? It was unclear to us what 15033-7 was referring to until we had reached the last paragraph in the first Results subsection. ▪ Why did the authors choose to recombine only the third CDR of the light chain together with the CDRs of the heavy chain? Please revise for clarity. ▪ In Figure 1D, the authors examined ACE2 binding to S-protein in the presence of three antibody candidates. It was a bit unclear to us how the signal was normalized – do the bars represent the percentage of the total signal seen with just control IgG? It may be useful to include a dotted line at Y = 1.0. o Figure 2: No issues. o Figure 3: No issues, though the class was unsure why the control antibody Trastuzumab was omitted in Figure 3C. o Figure 4: The class noted that this figure favoured Fab-IgG heavily. Are there representative images for IgG-Fab binding to the viral protein? Similarly, the diagrams all show the viral protein in the 'up' conformation. We were very keen to see how these interactions might change in the 'down' conformation'. o Figure 5: No issues. Are specific analyses performed using methods that are consistent with answering the specific question? o We had reservations about the alanine scanning mutagenesis experiment described in Figure 5. We understand this technique has been used to mimic viral escape mutations, however some of the mutations modelled would have required a change in two nucleotides (e.g. in Phe486). ● Is there the appropriate technical expertise in the collection and analysis of data presented? o Yes, ample technical expertise was demonstrated. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o N/A – there are no statistics featured in the manuscript. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o As mentioned above, we would have appreciated opportunities to assess affinities, potencies, and biophysical properties of existing antibodies against SARS-CoV2 to properly contextualize the results. 2. Quality: Completeness (1–3 scale) SCORE = 2 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o The data broadly support the conclusions made in the title and abstract o The class expressed some doubts about the thermostability results presented in supplemental figures. Some of the antibody candidates do not look as thermostable as Trastuzumab. Would this be worth addressing in the Discussion? ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o The authors may wish to examine antibody cytotoxicity. Non-cytotoxic concentrations should be used in their in vitro experiment (i.e. Figure 5), to exclude the possibility that the decrease in viral infection was due to treatment-related cell death. o As mentioned above, we had reservations about the thermostability of the synthetic antibodies compared to Trastuzumab. 3. Quality: Reproducibility (1–3 scale) SCORE = 2 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o It was unclear to us whether some of the figures showed results from two representative samples or whether only two replicates were performed in total per cohort (e.g. Figure 5A). o We could not find where in the manuscript the authors had specified the total number of biological and technical replicates performed per experiment. ● Is there sufficient raw data presented to assess rigor of the analysis? o We thought it would be useful for readers to examine the viral infection assay in greater detail. Can the authors provide the raw data for Figure 5B (in RLU) as supplemental information? o Line 133 mentions that 15033-7 saw significantly improved avidity compared to the other sixteen mutant 15033 antibodies, but we couldn't locate the data that supports this conclusion. o Lines 121-124 in the manuscript also mention that antibody 15033 "exhibited the highest avidity" compared against 15031 and 15032. Can the authors include this data in Table 1 or perhaps in a Supplemental Table? Are methods for experimentation and analysis adequately outlined to permit reproducibility? o It was unclear to us where the Fab sequences the authors used to construct their phage-display library were sourced. The authors reference a 2013 paper that describes how to construct such a library, however the manuscript does not describe how the sequences were obtained or where they were deposited. Could the authors provide any clarification in the Methods? o There is no mention in the Methods re: how the CDRs of the heavy and light chain regions were recombined in the 15033 antibody. The authors call this library of mutant antibodies the "light chain library" – was this library only created from swapping out CDRs in the light chain and not the heavy chain? o How were full-length IgG1 molecules created from the antibody fragments? We could not find how this was done in the Methods ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 2 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o The authors talk about vaccine hesitancy and they note that "how well they will be adopted by the public at large remains an unknown". However, there are vaccine hesitancy reports that include surveys on COVID-19 vaccines that the authors could cite and discuss: ▪ StatsCan Survey: https://www150.statcan.gc.ca/n1/pub/45-28-0001/2020001/article/00073-eng.htm ▪ Nature Medicine: https://www.nature.com/articles/s41591-020-1124-9. o The authors may wish to discuss some relevant COVID19 variants such as the South African and Brazilian variants in the context of their viral escape mutation experiment (Figure 5). ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o Are there any relevant papers that discuss how valency can affect an antibody's avidity? Are these two qualities related and complementary or is there a corresponding trade-off that the authors could discuss? o Our class also discussed how we would like the authors to include background and/or insight into the feasibility and challenges of delivering synthetic abs into a physiological system (i.e. animal model, people). Are there any issues with immunogenicity and lower half-lives (which was partially addressed in Figure 1E)? ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant bases for decisions. o Generally, the manuscript was written clearly and concisely. o We recommend a colour-coded legend for Figure 2 so readers will not have to flip back to the Figure Legends to understand the annotations. o In Figures 3B/D the colours for the lines are hard to resolve from one another, most especially the yellow lines against the white background or where the red lines are superimposed on top of the yellow lines. o It was unclear what the solid black lines that are superimposed on the coloured lines in Figure 3D represent. o There is a typo in the caption for Figure 3D (6.7 nM vs 67 nM in the Methods section) o We noticed that line 274 mentions 44 VLP variants, but lines 276-277 suggests that 49 variants were created. Is this a typo? MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.5 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. o The class agreed that the most interesting finding in this manuscript is the fabrication of the synthetic antibody and the subsequent workflow for engineering enhanced performance against SARS-CoV-2. o Owing to the present pandemic and the media attention given to the safety of proposed and existing treatments for COVID19, the class agreed that this study is interesting and important, and can be readily understood by both the public and the broader scientific community. ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o This manuscript outlines an antibody optimization workflow that can be readily followed and reproduced by other teams. If the antibodies arising from this pipeline work just as well in vivo as they did in vitro, then this paper is an important advance for the field. o The class agreed that this approach could be broadly applied to other viral infections. o The antibodies generated for this study were sourced from a library described in Persson, et al. (2013). Some students thought it challenging to consider the extensibility of this present manuscript without a more thorough description of this library in the Discussion 2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). o N/A – antibody efficacies were only evaluated in an in vitro model. o Though this is an N/A score, the class would like to note that we are very keen to see how well these synthetic antibodies perform in an in vivo infection model.

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/4768681. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Horizontal and vertical transmission of transgenerational memories via the Cer1 transposon. Rebecca S. Moore, Rachel Kaletsky, Chen Lesnik, Vanessa Cota, Edith Blackman, Lance R. Parsons, Zemer Gitai, Coleen T. Murphy. bioRxiv 2020.12.28.424563; doi: https://doi.org/10.1101/2020.12.28.424563 We will adhere to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029. SUMMARY: C. elegans can learn to avoid pathogenic P. aeruginosa by processing bacterial small RNAs in intestinal epithelial cells, transmitting this genetic information to neurons to achieve avoidance behavior and germ cells to vertically transmit avoidance behavior to ~4 future generations. Here, Dr. Coleen Murphy's team demonstrate that this genetic information is encapsulated by capsids encoded by the Cer1 retrotransposon. In addition to vertical transmission, Cer1 capsids are also required for horizontal transmission to naïve animals. Thus, C. elegans has usurped a retrotransposon to enable transgenerational immune memory. OVERALL ASSESSMENT: STRENGTHS: The preprint is clearly and concisely written, and the data is clearly presented. It addresses a fascinating topic of broad interest. In general, the experimental approaches are sound and the author's conclusions are well-supported by the data. WEAKNESSES: The manuscript could be further strengthened by conducting biochemical/genetic analysis of VLPs (which is likely to be quite technically challenging, as discussed by the authors). The authors should also consider placing these findings in the context of recent studies from Maria-Carla Saleh's lab on vertical transmission of immune memory in flies, as there could be some relevant parallels between these two models. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY) 1. Quality: Experiments (1–3 scale) SCORE = 1.5 ● Figure by figure, do experiments, as performed, have the proper controls? [Note: in this section, the class discusses proper controls, but also uses the 'figure-by-figure' opportunity to discuss rationale and approaches] o Figure 1: We didn't find any issues with the controls, although there was some discussion about the rationale for using the F2 generation as substrate for horizontal transfer experiments. Why F2 and not one (or a few) of the other generations? Is it a pragmatic choice, to avoid longer or larger experiments that might test multiple generations? ▪ The students noted the difference in aversion behavior between progeny that are lawn trained vs P11 RNA trained (compare Fig 1 B and 1C). Could the authors comment on these differences? It would have been nice to have seen F2 aversion from lawn-trained worms, similar to the experiment shown in Fig 1C using P11. ▪ In Figure 1K the authors tested if the worms could also learn to avoid P. florescens and S. marcescens, but the rationale for the choice of these bacteria specifically was unclear. We know that C. elegans avoids P. aeruginosabecause the bacteria can cause disease; would the worm have a reason to avoid P florescens and S marcescens if they do not cause disease? ▪ The authors could provide more detailed methodology about the Choice Index assay. o Figure 2: There was some discussion about the use of electron microscopy to identify VLPs. It is likely that Fraction 6 is quite heterogeneous, and additional particles beyond VLPs may be present, including non-VLP particles that might be expected to stain with an anti-Cer1 antibody. Could the authors address this possibility or cover it in the Discussion? o Figure 3: There was no issue with controls, but the class thought that the logical leap from Figure 2 to focus specifically on Cer1 was not well explained. o Figure 4: OK o Figure 5: Panel A is confusing without either a visual schematic of when RNAi is administered or a better explanation within the figure legend. o Figure 6: OK o Figure 7: OK (but this figure prompted a discussion about matricide/cannibalism and potential for horizontal transfer of immune memory in nature). ● Are specific analyses performed using methods that are consistent with answering the specific question? o The experimental approaches were generally sound. ● Is there the appropriate technical expertise in the collection and analysis of data presented? o Appropriate technical expertise was demonstrated. ● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons? o There is no mention of what statistical tests were performed to support the data, figure by figure. The methods mentions that Prism8 was used for statistics but does not say what tests were performed. This should be addressed. ● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship. o The experimental foundations of this study are generally solid. The authors cited the proper literature and presented strong data that was sufficient to support their conclusions. 2. Quality: Completeness (1–3 scale) SCORE = 1 ● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems. o Generally, the data supports the conclusions as stated in the title and abstract. o The class had some reservations about the evidence provided for a functional role for Cer1 VLPs specifically rather than other types of extracellular vesicles that might also contain Cer1 antigen. ● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. o N/A 3. Quality: Reproducibility (1–3 scale) SCORE = 1 ● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? o Sufficient numbers of animals and biological and technical replicates were performed to provide a high-confidence dataset. ● Is there sufficient raw data presented to assess rigor of the analysis? o Yes Are methods for experimentation and analysis adequately outlined to permit reproducibility? o The methods are generally clearly described (as judged by this group of non-experts). The authors may consider providing clearer descriptions of statistical tests, as mentioned above. ● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed? o N/A 4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5 ● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? o Yes ● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work? o The authors may consider discussing this recent paper, which describes mechanisms of RNA-mediated vertical transfer of immune memory in Drosophila melanogaster and Aedes egyptii. ▪ Mondotte JA, Gausson V, Frangeul L, Suzuki Y, Vazeille M, Mongelli V, Blanc H, Failloux AB, Saleh MC. Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects. Cell Rep. 2020 Dec 15;33(11):108506. doi: 10.1016/j.celrep.2020.108506. PMID: 33326778; PMCID: PMC7758158. o No one in the class has first-hand experience with C. elegans research, so we would have benefitted from additional information about the frequency and circumstances of matricide/cannibalism in C. elegans or other roundworms. This would provide helpful context about opportunities for horizontal transfer of immune memory between worms in nature. ● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions. o The writing was clear and concise. The lean writing style did not hinder reader comprehension. We really liked the first sentence of the abstract, which puts the study in much broader context and 'hooks' the reader. 'Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within." o Consistency in Choice Index y-axis labelling throughout would be helpful. o Clear figure labeling to indicate that OP50 is E. coli would be helpful (there was some confusion about that) o In Figure 1J, the students were confused about the 'N2' label. o In Figure 7, the students suggested that consistency in labeling either 'Hawaiian' or 'CB4856'. MORE SUBJECTIVE CRITERIA (IMPACT) 1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1 ● A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field. o The key finding relates to the Cer1 capsids serving as the vehicle for both vertical and horizontal transfer of immune memory. The concept of the helpful capsids could be understood by a layperson because they are conceptually simple (i.e. a pathogen-derived 'box' that has been usurped by the host and now helps to transmit immune memories). ● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here. o There was broad agreement that this manuscript represents a considerable advance without further extension. o The authors draw a parallel between their system and the Arc retrotransposon of flies that supports the plasticity of the neuromuscular junction. This prompted additional discussions about other relevant models like the mechanisms of RNA-mediated vertical transfer of immune memory in Drosophila melanogaster and Aedes egyptii, as described above. o If 'helpful' retrotransposons involved in immunity are subsequently discovered in other eukaryotes, these studies should be considered foundational. o There was some discussion about the proposed RNA-seq experiment on VLPs. The class acknowledged the technical challenges of generating sufficient VLP material for an RNA-seq experiment, but some students thought that this experiment may be required to satisfy peer reviewers. 2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A ● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? ● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). o N/A o Even though this is clearly an 'N/A' situation, the class discussed the paper in the context of the Drosophila Arc retrotransposon, and remains intrigued about whether retrotransposons may perform similar functions in other eukaryotes.

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:Souvik Bhattacharyya, David M. Walker, Rasika M. Harshey. Necrosignaling: Cell death triggers antibiotic survival pathways in bacterial swarms. BioRxiv 2020.02.26.966986; doi: https://doi.org/10.1101/2020.02.26.966986We adhered to the Universal Principled (UP) Review guidelines proposed in:Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. https://doi.org/10.1016/j.cell.2019.11.029SUMMARY: In this manuscript, Bhattacharyya, et al. investigated the mechanism of how the death of a sub-population of swarming bacterial cells increased survival of the remaining cells. They called this phenomenon STRIVE (swarming with temporary resistance in various environments). Using E. coli and several other bacteria, the authors identified a candidate STRIVE factor called AcrA that was released by dead cells. They also demonstrated that the outer membrane protein TolC on the live cells is involved in the STRIVE response, possibly interacting with AcrA released from dead cells. Microscopy studies of mutant bacteria suggested that AcrA binds to TolC on the exterior of live cells and triggers STRIVE. Finally, the authors investigated the gene expression profile of the swarming cells and proposed that increased AcrA-dependent efflux and ROS catabolism, and reduced cell permeability, are potential mechanisms of STRIVE. In summary, this work reveals that AcrA is an enhancing factor of STRIVE that interacts with TolC to stimulate the reprogramming of live cells.OVERALL ASSESSMENT: STRENGTHS: · The work provides a new insight of how the dead subpopulation contributes to swarming cell resistance. The findings are novel and impactful and merit further exploration.· The authors thoroughly investigated the mechanisms of necrosignaling/STRIVE using a suite of complementary approaches.WEAKNESSES: · The reader needs more thorough Introduction and Discussion sections to put these new discoveries (necrosignaling/STRIVE) in proper context in the field. These sections are quite underdeveloped. · Modifications in a few figures and more detailed methodology will help the reader interpret the data.· Statistical analyses are not well described for most data.DETAILED U.P. ASSESSMENT ("1" represents the highest quality)OBJECTIVE CRITERIA (QUALITY): 1. Quality: Experiments (1-3 scale) SCORE = 1.5 Figure by Figure, do experiments, as performed, have the proper controls? · Fig 1: This figure is properly controlled, but the readability of this section of the Results is poor, because the reader must flip back-and-forth between the Supplemental figures and Figure 1 to properly understand the experimental approach and the results. The authors should consider including parts of Figs S1 and S3, as well as a smaller version of the cartoon in Figure S2, to help the reader understand method and important findings. The current compressed format that relegates important data to the supplement, does not serve the reader well.· Fig 2: The findings displayed in Fig. 2C would be stronger if a wild-type control was included.Are specific analyses performed using methods that are consistent with answering the specific question? Is there the appropriate technical expertise in the collection and analysis of data presented?· YesIs there the appropriate technical expertise in the collection and analysis of data presented? · Fig S1: We think that the relative susceptibility of swarm cells at early times and low doses of antibiotic is quite subtle, and unless the reader consults the supplemental data, they might misunderstand this subtle finding. The description of this finding in the Results text should acknowledge the modest difference in susceptibility. Moreover, we wonder whether this small difference could be an artefact resulting from the collection method rather than true antibiotic susceptibility. However, this experiment was incompletely described in the methods, which makes it difficult to interpret. Appropriate statistical tests should be performed on the data in Fig. S1 to determine whether these modest differences are significant or not.Do analyses use the best possible (most unambiguous) available methods, quantified via appropriate statistical comparisons?· Fig 3c & 3d: The slopes of these curves are shown, but statistical analyses would be helpful in determining whether true significant differences are achieved. · When error bars and p values are presented, the type of error (SD or SEM) and statistical method used should be described.Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least 2 examples in the literature of such results. To address this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study. · The experiments are consistent with previous findings that swarming cells develop adaptive antibiotic resistance.2. Quality: Completeness (1-3 scale) SCORE = 1.5Does the collection of experiments and associated analysis of data support the proposed title/abstract-level conclusions? Typically, the major (title or abstract level) conclusions are expected to be supported by at least two experimental systems.· Most of the major conclusions are supported by the experimental results. The only egregious unsupported statement is on line 38: "...our findings may also be relevant to chemotherapy-resistant cancers". The link to cancer chemotherapy resistance is not made clear in the manuscript and is not directly addressed by any of the data. It should be removed. Are there experiments or analyses that have not been performed, but if "true" would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion/abstract that is clearly defensible with the experiments as presented, and one solution to 'completeness' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.· We think that further description of the RNA-seq analysis is needed to clarify whether functional categories were significantly different overall or if other non-related categories were also significantly different. It is especially important that the authors reveal how they identified the gene families shown: were all gene families of porins/ROS metabolism/efflux pumps shown, or were the families shown selected from a group?· Despite the attempt to identify critical residues for AcrA-TolC interaction, it is unclear how this extracellular interaction contributes to increased efflux and regulation of other signaling pathways, since AcrA usually acts as a periplasmic protein. As a major conclusion of this manuscript, the authors should at least discuss what they think is the most likely mechanism in the Discussion, or state that it is still unclear how this mechanism works.· It would be nice to have a piece of data like an SDS-PAGE of the purified AcrA (could be placed in the Data Supplement) to show the quality of their purification, as any other residual E. coli proteins could influence the results.· The authors should explain how they chose the antibiotics used in the study. In addition, it is unclear why Kan50 was used in Fig 1b and Kan70 in Fig 1d.3. Quality: Reproducibility (1-3 scale) SCORE = 2 Figure by Figure, were experiments repeated per a standard of 3x repeats or 5 mice/cohort etc.?· Except for the RNA-Seq analysis, most figures and methods do not indicate how many repeats were performed, especially for those that had error bars and p values like Fig 1a, 3b, & 3d. Is there sufficient "raw data" presented to assess rigor of the analysis? · Yes. Are methods for experimentation and analysis adequately outlined to permit reproducibility?· Methods for the Kanamycin survival curves and MIC determination (Fig S1) were not described.· More details are needed on how the RNA-seq data was analyzed. We also suggest that the authors deposit the code that was used to create the simulations and do the RNA-seq analysis in public repository such as github.4. Quality: Scholarship (1-3 scale), generally not the basis for acceptance/rejection: SCORE = 3Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?· Yes. In the Discussion the authors mention how their finding is different from previous studies that reported other functions of cell lysis.Has the author cited and/or discussed the important works that are consistent with their conclusion and which a reader should be especially familiar when considering the work?· The Introduction and Discussion should be expanded to help readers appreciate previous findings in the field and to put the current work in proper context of larger themes like chemotaxis and quorum sensing.Specific (helpful) comments on grammar/diction, paper structure or data presentation (e.g. change a graph style or color scheme) go in this section, but scores in this area not to be significant bases for decisions.· The switching between "STRIVE" and "SR" is rather confusing. It would be clearer to use just one or the other consistently throughout the text.· The resolution of pictures in the main figures should be increased if possible (this may be an issue with the BioRxiv uploader).o Fig 2c: Mutated amino acids are not visible.o Fig 2e: Panel FM 1-43 - deltaTolC/pTolC, looks like cellular debris; it is difficult to see any bacterial morphology, and we suggest that this image should be replaced by a clearer picture if possible. Many figures are missing figure keys. · The heatmaps in each figure should all include figure keys so that the reader doesn't have to find this information in the figure legend. Fig 1f and S1 should also have a figure key.· Fig 1a: It would be clearer to directly label "1" and "2" as "homogeneous" and "heterogeneous".· Fig 2c: We suggest changing numbers I, II, III and IV (in diagram) to TolC mutants, WT AcrA, WT AcrA, AcrA mutants, respectively.· Fig S12: The use of color (Blue, SR+; Maroon, SR-) is confusing as it appears different from the other figure legends.· Although it is described in the Methods section, we suggest that the authors describe how they prepared the samples for the MS/MS analysis in the Results section (lines 111-113). This would help the readers understand the massive work performed to identify the final 5 common proteins.· Some lines of text are grey in colour instead of black (lines 48, 49, 427).· The short Introduction, short Discussion, and the extensive Supplementary figures make the manuscript very difficult to read.· Grammatical error:o Line 64: "The aim of present study" should be changed to "The aim of the present study". MORE SUBJECTIVE CRITERIA (IMPACT):1. Impact: Novelty/Fundamental and Broad Interest (1-4 scale) SCORE = 2A score here should be accompanied by a statement delineating the most interesting/important conceptual finding(s), as they stand right now with the current scope of the paper. A '1' would be expected to be understood for the importance by a layperson but would also be of top interest (will have lasting impact) on the field. How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Table 2). Citations (figure by figure) are essential here. · We think this work is very interesting in terms of the overall rationale and experimental design. That being said, more background and discussion should be provided to assess its novelty and impact on the field. Specifically, (i) how necrosignaling differs from previously known chemotaxis/quorum sensing mechanisms, and (ii) whether chemotaxis toward products from the lysis of other bacteria in the same species has been described.2. Impact: Extensibility (1-4 scale) SCORE = N/AHas an initial result (e.g. of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g. in animals, or in a human cohort)? This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g. 'repeat a mouse-based experiment in humans') and difficulty that merely confirm, but do not extend. (see Bad Behaviors, Table 2).· More rationale is needed to determine extensibility. Specifically, the reader needs more discussion about how STRIVE-induced adaptive resistance differs from that induced by biofilms and how it relates to what we already know about quorum-sensing and chemotaxis.· The necrosignaling phenomenon could be further investigated in other bacterial species such as Gram-positive bacteria like Listeria monocytogenes, Listeria innocua, Proteus vulgaris, Clostridium spp., etc., and the Discussion could describe potential extensibility of this aspect.

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:Gordon et al. A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing. BioRxiv doi: https://doi.org/10.1101/2020.03.22.002386.We will adhere to the Universal Principled (UP) Review guidelines proposed in:Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. https://doi.org/10.1016/j.cell.2019.11.029SUMMARY: The current COVID-19 pandemic has motivated a worldwide effort to discover and develop vaccines and antivirals for SARS-CoV-2. In this manuscript, Gordon et al. aim report on the protein-protein interactome between SARS-CoV-2 proteins and human proteins to identify potential candidate drugs and their target human proteins. The authors used an affinity purification mass spectrometry (AP-MS) approach, involving 26 Strep-tagged viral proteins expressed in human HEK293T cells. Cell lysates were affinity purified and subjected to mass spectrometry. 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs) were found. Among these, 67 human proteins were known to be targeted by existing drugs. As expected, SARS-CoV-2 proteins are connected to a wide array of biological processes. This study provides a methodology that can be adopted to study of the interactome of other viruses. Importantly, Gordon et al. provide valuable data that can become the foundation for further mechanistic studies and, ultimately, clinical trials. OVERALL ASSESSMENTSTRENGTHS: This manuscript is the result of a rapid response from a large international team of researchers; their report provides a strong starting point for future drug development and drug-repurposing. Confidence in the interactome is bolstered by agreement with previous findings of viral targeting of host proteins involved in key nodes of control, both from previous coronavirus studies and other viruses studied by this group. Overall, this manuscript is well written and the figures are described in appropriate detail. Also, all raw data is available for other researchers to readily use. WEAKNESSES: The primary weakness of the study are that the authors did not confirm any protein-protein interactions using standard confirmatory methods like co-immunoprecipitation. This would obviously involve a great deal of labor, but some key interactions that are being used to inform selection of candidate FDA-approved drugs should be confirmed. The manuscript would also be strengthened by testing some high-priority candidate drugs in an in vitro SARS-CoV-2 infection assay like plaque assay or TCID50. DETAILED UP ASSESSMENT ("1" represents the highest quality)OBJECTIVE CRITERIA (QUALITY)1. Quality: Experiments (1–3 scale) SCORE = 1Figure by figure, do experiments, as performed, have the proper controls?o Proper controls were employed in each of the figures. o We were impressed with the Extended Data Figure 1, which demonstrates high correlation of the replicates.Are specific analyses performed using methods that are consistent with answering the specific question?o Yes.Is there the appropriate technical expertise in the collection and analysis of data presented?o Yes.Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?o The clustering method seems to be in line with others.o The GO method (clusterProfiler package) is commonly used for this kind of analysis.o We suggest uploading the code to a public repository. Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.o Controls and experiments used here are in line with field standards for protein-protein interactions.2. Quality: Completeness (1–3 scale) SCORE = 2Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.o This study aimed to find potential drug targets and they used one approach (AP-MS) and one system (HEK293T cells). However, the dataset appears to be robust and the authors used a bioinformatics approach to validate their findings in the context of previously published data (Fig 2c-e). o Nevertheless, we consider that the limitations of using HEK293T over a lung cell line should be further clarified/discussed.o This paper has a strong bioinformatics component but lacks confirmatory biochemical analysis of the interactions. o A few protein targets could have been tested in vitro to evaluate the relevance of these interactions to viral life cycle (e.g. entry). o We suggest that the authors could re-evaluate title and maybe change it to "A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Potential Drug Targets"Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.o The results support the conclusions. However, the lack of biochemical confirmation of the interactions and their role in SARS-CoV-2 infection needs to be clearly stated in the Discussion. We are worried that these druggable targets that haven't been validated in any way could be taken as definitive treatment alternatives by the lay public and medical professionals tempted by off-label use. 3. Quality: Reproducibility (1–3 scale) SCORE = 1.5Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?o Yes. Is there sufficient raw data presented to assess rigor of the analysis?o All mass spectrometry raw data and search results files have been deposited to the ProteomeXchange Consortium.o The supplementary tables also include extensive raw data, which will be useful for the field Are methods for experimentation and analysis adequately outlined to permit reproducibility?o Methods for figure 2c are not in the main text (methods section). We suggest to move methods for Supl. fig. 4-5 to the main text.o In methods, the authors should clarify which constructs have N- vs C- terminal tagso We suggest more detail is included for the chemoinformatics analysis. Also, approach 2) "a target- and pathway-specific literature search, drawing on specialist knowledge within our group" needs to be better described for the reader. Such specialist knowledge, if not properly described, may limit the ability of others to confirm the work.4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?o In the Supplementary Discussion, the authors clearly state the possible limitations of the use of homologous proteins from other coronaviruses to infer the SARS-CoV-2 protein and gene functions. Thus, we suggest this disclaimer could also be made in the main Discussion. Despite this limitation, we think the review of the principle interactions for each bait (Sup. Discussion) is very valuable for the scientific community at this point.Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?o Discussion of data from previously published coronaviruses interactomes could be improved. E.g. citing articles like this one https://elifesciences.org/articles/42037. Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant bases for decisions.o Abstract and Introduction: Mentions number of infected since the end of 2019, but does not give a "current" as of written date. It would be better to have written: "290,000 people infected from the onset (as of DATE)." This will significantly help readers who haven't been following numbers day-by-day especially as this is a developing situation.o Fig 1c. Authors could refer to the other bands that did not match the expected size of construct. Is this the product of protein degradation/aggregation?o 1d: are the beads coated with antibodies or a modified streptavidin? Please modify text to clarify this for the reader. o Sup. Fig. 1: labels are pixelated, but might be a submission issue on the BioRxiv site. o Fig 2c: we suggest adding a figure with all 29 tissues tested to the supplementary material. o Fig 2d: clarify the biological relevance of this figure and why were you expecting a positive correlation? Provide rationale/discuss this figure in greater detail. Otherwise we suggest moving it to supplementary material. o Fig 2e: we suggest moving the discussion points about this figure from the supplementary material and place it in the main text, to help the reader understand the importance of this figure. o Sup. Fig 5: we suggest, if possible, moving this figure to the main text because it shows a lot of possible important interacting proteins and the drugs that could target these proteins.o Sup. Fig 6: we think that this figure might be beyond the scope of this paper and is not enough to support that the interactome (332 proteins) has reduced genetic variation in human populations. We suggest that this paragraph should be moved to the Supplementary Discussion. Also, a T-test might not be appropriate for testing differences in loss of function. We suggest the authors use a Wilcoxon test as the data distribution did not look normal (especially for the loss of function data).o Fig 4: This figure is overcrowded. We suggest to simplify the figure or split it in two. Also, in the main text, subpanels are not specified (eg: Fig 4a-i)o Fig 5: yellow levels are difficult to see.o When downloading the manuscript some lines have single spacing and some have 1.5 line spacing (pages 5, 14), might be an issue of the bioRxiv site.MORE SUBJECTIVE CRITERIA (IMPACT)1. Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 2A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.● Very relevant and broad interest topic. ● Opens up opportunities for other labs to test specific therapeutics and investigate host targets and or pathways identified in the paper for potential treatments.How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.o We think given the current pandemic, this paper will have a huge impact as it is, without any extended experiments. However, if functional data is included the impact will be higher. 2. Impact: Extensibility (1–4 or N/A scale) SCORE = NAHas an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/3718438. We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint: Spreading of a virulence lipid into host membranes promotes mycobacterial pathogenesis CJ Cambier, Steven Banik, Joseph A. Buonomo, Carolyn Bertozzi bioRxiv 845081; doi: https://doi.org/10.1101/845081 We adhered to the Universal Principled (UP) Review guidelines proposed in: Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. https://doi.org/10.1016/j.cell.2019.11.029 SUMMARY: Cambier CJ, et al. investigated the mechanism by which a virulence lipid on the outer mycomembrane (OM) of Mycobacterium marinum prevents host immune activation. They report on a system for extracting lipids from the OM, and also for reconstitution of OM lipids. Using this technique, the authors confirmed that the virulence lipid, phthiocerol dimycocerosate (PDIM), is required for avoiding host toll-like-receptor (TLR)-dependent immune responses to aid bacterial infection. The authors describe a novel method for labelling specific lipids on the OM. Using this approach, the authors demonstrated that PDIM spreads away from the M. marinum OM and into the membranes of nearby zebrafish host cells. By fixing PDIM to the OM to prevent spreading, they demonstrate that this spreading phenotype is required to avoid the host TLR-dependent immune response. Overall, we thought this was an excellent paper. Although we were confused about several findings, we believe the authors could likely address our main concerns by including clarifying statements in the text. OVERALL ASSESSMENT: STRENGTHS: Overall, we thought that a clear rationale was provided for each experiment. The manuscript introduced interesting new techniques like lipid reconstitution and labelling. New insights into PDIM spreading into host membranes could be valuable for better understanding Mycobacterium pathogenesis in humans and warrants further research. WEAKNESSES: Several results were difficult to interpret and could be misleading without further clarification. In particular, distinguishing between levels of TLR-independent and TLR-dependent macrophage activation was confusing. In at least one instance, a control experiment that would greatly aid interpretation is missing. DETAILED U.P. ASSESSMENT: OBJECTIVE CRITERIA (QUALITY): 1. Quality: Experiments (1-3 scale) SCORE = 1.5 Figure by Figure, do experiments, as performed, have the proper controls? · In general, we thought the authors did an excellent job of including appropriate controls. For instance, we were convinced by their hydroxylamine control experiments in Figure 4E that the fluorescent modifications themselves were unrelated to the in vivo spreading of PDIM that they report. · However, in Figure 2C the bacterial volumes for both M. marinum strains should also be reported in MyD88-deficient zebrafish. This would allow us to make clearer comparisons to the findings in Figure 2D. Are specific analyses performed using methods that are consistent with answering the specific question? Is there the appropriate technical expertise in the collection and analysis of data presented? · Overall the authors used the appropriate methods for answering their specific questions (although we lack some relevant expertise, as noted below). Our issues are not with these methods, but instead with interpretation of results. However, we did have issues with several analyses, which may be resolved by adding clarification in the main text. · We were confused about the results reported in Figure 2D regarding macrophage recruitment by bacteria. If WT lipids are required to circumvent the TLR response, we believe that the expected result would be low macrophage recruitment for the WT lipid treatment regardless of whether the zebrafish are MyD88-deficient. Instead, the recruitment levels seem similar to the result with mmpL7 knockout lipids when MyD88 is present. At the very least, this finding requires more explanation in the text. · Most of the in vivo spreading experiments are focused on spreading specifically to macrophages. However, azido-DIM spreading to epithelial cells occurs in the absence of macrophages (Figure 4F and G). Although the authors do point this out, it remains unclear whether there is a difference in affinity between macrophages and epithelial cells. · For Figure 6E, we were also confused why the WT Live samples had such high macrophage recruitment. Again, this result seems at odds with the conclusion that WT lipids enable the bacterium to down-regulate TLR-dependent activation of macrophages. The confusion here may arise due to the difficulty in distinguishing macrophages recruited from TLR-dependent and TLR-independent signaling. Again, at the very least this distinction should be clarified in the main text. In particular, the authors should emphasize that the data in Figure 6E (and Figure 2D) is consistent only with PDIM-deficient bacteria or bacteria unable to spread PDIM onto host membranes to require TLR signaling to recruit macrophages. Based on the macrophage recruitment findings alone it is not convincing that these cells are detrimental. Also, the authors should emphasize that macrophage recruitment is high irrespective of lipid composition. Is there the appropriate technical expertise in the collection and analysis of data presented? · To the best of our knowledge the authors have the appropriate expertise. However, we should note that many of the techniques in this study, and particularly the chemistry work, is outside our own expertise. Do analyses use the best possible (most unambiguous) available methods, quantified via appropriate statistical comparisons? · We did find certain approaches ambiguous (see above points), which should be clarified. · Many of the comparisons from Figure 2 onwards did not include any statistical tests. We do not anticipate that the key findings of this paper will change after including statistical tests, but nonetheless these should be included to make the results more convincing. In addition, significance stars should be indicated on the figures and the authors should describe all statistical tests they perform. · Several times in the text the authors mention their result is representative of three separate experiments. Based on this wording, it is unclear whether all reported differences were reproducible across separate biological replicates. If there were no differences in interpretations across different experiments this should be clearly stated. Alternatively, if there was a lot of variation across experiments, then this additional data should be included in the manuscript (perhaps as supplementary figures depending on the extent of disagreement). Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least 2 examples in the literature of such results. To address this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study. · The key findings of this paper are consistent with other work in this area. 2. Quality: Completeness (1-3 scale) SCORE = 1 Does the collection of experiments and associated analysis of data support the proposed title/abstract-level conclusions? Typically, the major (title or abstract level) conclusions are expected to be supported by at least two experimental systems. · The authors' fluorescent labelling approach convincingly showed that PDIM spreads into host membranes. · PDIM spreading was shown to be required for the TLR-dependent activation of macrophages (Figure 6E), although we are unclear why the numbers of macrophages would be so similar when recruited through a TLR-independent route (i.e. for the Live samples). · They also convincingly show that PDIM spreading into host membranes promotes pathogenesis, based on the observed increases in bacterial volume (especially in Figure 6F). Are there experiments or analyses that have not been performed, but if "true" would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion/abstract that is clearly defensible with the experiments as presented, and one solution to 'completeness' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section. · Again, we believe further clarification is needed to discuss the high levels of macrophage recruitment due to a TLR-independent process. If a clear explanation cannot be given, then there could be issues with how some of the results in this paper are interpreted. However, we think the main conclusions of the article are robust. 3. Quality: Reproducibility (1-3 scale) SCORE = 1 Figure by Figure, were experiments repeated per a standard of 3x repeats or 5 mice/cohort etc.? · The sample sizes of all experiments presented were sufficient. Is there sufficient "raw data" presented to assess rigor of the analysis? · Yes, the authors have done a great job of presenting a lot of data with sufficient controls to judge the data. Are methods for experimentation and analysis adequately outlined to permit reproducibility? · Yes, we have no concerns with how the methods are described. If a 'discovery' dataset is used, has a 'validation' cohort been assessed and/or has the issue of false-discovery been addressed? · N/A. 4. Quality: Scholarship (1-3 scale), generally not the basis for acceptance/rejection: SCORE = 1 Has the author cited and discussed the merits of the relevant data that would argue against their conclusion? · No, the authors do not specifically discuss such relevant data. We do not have any specific recommendations for this point except that expanding the discussion would be useful and could aid interpretation of individual results (if that is permitted in the format). Has the author cited and/or discussed the important works that are consistent with their conclusion and which a reader should be especially familiar when considering the work? · The authors clearly describe similar work in the introduction that demonstrated that PDIM is required for Mycobacterium marinum to avoid the TLR signaling response in zebrafish. Their work clearly fits in a larger story that includes these studies as well. · The authors do discuss relevant work. In particular, they mention that the in vivo work complements a similar study that demonstrated that PDIM incorporates into host membranes in a macrophage in vitro model. Specific (helpful) comments on grammar/diction, paper structure or data presentation (e.g. change a graph style or color scheme) go in this section, but scores in this area not to be significant bases for decisions. · We noticed that a lot of introductory material is presented in the results section (e.g. the first paragraph of the results). We recommend that this information be moved to the introduction. · Write out (and indicate) acronyms in figure legends, for instance: o Indicate that dpi stands for days post infection in Figure 1 legend o Write out TLC, PDIM, and PNDIM in Figure 2 legend · We recommend that "wondered" be changed to "investigated" or re-worded to be "hypothesized" where appropriate (occurs several times - e.g. on page 3) · "Clickable" should be clearly defined upon first usage · Page 1: Change "A result" to "As a result" · Page 3: Change "do not repopulate the" to "do not repopulate in the" · We recommend that "where" be replaced with "in which" in certain places: o "we used the transgenic zebrafish line [...] where macrophages express [...]" o "PFA+GA-treated bacteria, where PDIM is no longer able to spread [...]" o "our group developed metabolic labeling strategies where unnatural metabolic precursors [...] are fed [...]"). · At several points the authors discuss that results indicate that the presence of lipids at the onset of infection are required for virulence. We found this language a little confusing because it implies that the experiments included post-infection lipid modification. We recommend that this is clarified. MORE SUBJECTIVE CRITERIA (IMPACT): 1. Impact: Novelty/Fundamental and Broad Interest (1-4 scale) SCORE = 2 A score here should be accompanied by a statement delineating the most interesting/important conceptual finding(s), as they stand right now with the current scope of the paper. A '1' would be expected to be understood for the importance by a layperson but would also be of top interest (will have lasting impact) on the field. · The key contributions are to confirm in an in vivo model that PDIM spreads to host membranes and is required for down-regulating the TLR-dependent activation of macrophages. · We also think the methods that the authors have developed will be valuable for future work in this area. · We do not anticipate that this paper will necessarily be of interest to a lay audience, but it would be of interest to a broad group of microbiologists and immunologists. How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Table 2). Citations (figure by figure) are essential here. These findings will be impactful without additional extension. The authors have performed numerous control experiments such as in Fig S1, Fig 1, and Fig 3 to convince us that their new methods are robust and reliable. In addition, the key results reported in Fig 6 will likely be of great interest to tuberculosis researchers. Of course, eventually extending this work into different model systems will be required to show that these results are important for understanding tuberculosis in humans. 2. Impact: Extensibility (1-4 scale) SCORE = N/A Has an initial result (e.g. of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g. in animals, or in a human cohort)? This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g. 'repeat a mouse-based experiment in humans') and difficulty that merely confirm, but do not extend. (see Bad Behaviors, Table 2). · Extending the work to explore the specific mechanism underlying the TLR response downregulation would be valuable. · In addition, this paper focused on zebrafish and it will be interesting to see how well the