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Nirmatrelvir, an oral antiviral targeting the 3CL protease of SARS-CoV-2, has been demonstrated to be clinically useful in reducing hospitalization or death due to COVID-191,2. However, as SARS-CoV-2 has evolved to become resistant to other therapeutic modalities3–9, there is a concern that the same could occur for nirmatrelvir. Here, we have examined this possibility by in vitro passaging of SARS-CoV-2 in increasing concentrations of nirmatrelvir using two independent approaches, including one on a large scale in 480 wells. Indeed, highly resistant viruses emerged from both, and their sequences revealed a multitude of 3CL protease mutations. In the experiment done at a larger scale with many replicates, 53 independent viral lineages were selected with mutations observed at 23 different residues of the enzyme. Yet, several common mutational pathways to nirmatrelvir resistance were preferred, with a majority of the viruses descending from T21I, P252L, or T304I as precursor mutations. Construction and analysis of 13 recombinant SARS-CoV-2 clones, each containing a unique mutation or a combination of mutations showed that the above precursor mutations only mediated low-level resistance, whereas greater resistance required accumulation of additional mutations. E166V mutation conferred the strongest resistance (~100-fold), but this mutation resulted in a loss of viral replicative fitness that was restored by compensatory changes such as L50F and T21I. Structural explanations are discussed for some of the mutations that are proximal to the drug-binding site, as well as cross-resistance or lack thereof to ensitrelvir, another clinically important 3CL protease inhibitor. Our findings indicate that SARS-CoV-2 resistance to nirmatrelvir does readily arise via multiple pathways in vitro, and the specific mutations observed herein form a strong foundation from which to study the mechanism of resistance in detail and to inform the design of next generation protease inhibitors.

SummarySARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) as the etiologic agent of COVID-19 (coronavirus disease 2019) has drastically altered life globally. Numerous efforts have been placed on the development of therapeutics to treat SARS-CoV-2 infection. One particular target is the 3CL protease (3CLpro), which holds promise as it is essential to the virus and highly conserved among coronaviruses, suggesting that it may be possible to find broad inhibitors that treat not just SARS-CoV-2 but other coronavirus infections as well. While the 3CL protease has been studied by many groups for SARS-CoV-2 and other coronaviruses, our understanding of its tolerance to mutations is limited, knowledge which is particularly important as 3CL protease inhibitors become utilized clinically. Here, we develop a yeast-based deep mutational scanning approach to systematically profile the activity of all possible single mutants of the SARS-CoV-2 3CLpro, and validate our results both in yeast and in authentic viruses. We reveal that the 3CLpro is highly malleable and is capable of tolerating mutations throughout the protein, including within the substrate binding pocket. Yet, we also identify specific residues that appear immutable for function of the protease, suggesting that these interactions may be novel targets for the design of future 3CLpro inhibitors. Finally, we utilize our screening results as a basis to identify E166V as a resistance-conferring mutation against the therapeutic 3CLpro inhibitor, nirmatrelvir, in clinical use. Collectively, the functional map presented herein may serve as a guide for further understanding of the biological properties of the 3CL protease and for drug development for current and future coronavirus pandemics.

SUMMARY IQGAP1 is a master regulator of many cellular processes, including intracellular vesicle trafficking and endocytosis. We show that depletion of IQGAP1 in a variety of cell types increases the release of HIV-1 infectious virions and that overexpression diminishes virion production, with neither affecting the early stages of infection. IQGAP1 negatively regulates the steady-state levels of HIV-1 Gag at the plasma membrane, the site of assembly. We establish that IQGAP1 interacts with both the nucleocapsid and p6 domains of Gag, and interaction with either domain is sufficient for its regulatory function. Finally, we demonstrate that IQGAP1 regulation is independent of HIV-1 Gag “late-domains” sequences required by the virus to recruit the cellular ESCRT machinery. Thus, we provide evidence that IQGAP1 is a negative regulatory factor inhibiting efficient budding of HIV-1 by reducing Gag accumulation at the plasma membrane., Graphical Abstract, In Brief IQGAP1 is a ubiquitously expressed master regulator of many cellular processes, including intracellular trafficking. Sabo et al. demonstrate that in a variety of cell types, IQGAP1 acts as a negative regulator of HIV-1 viral particle release by reducing accumulation of the Gag viral structural protein at the plasma membrane.

Significance Invading viral DNAs constitute a high risk for the infected cell and are high-profile targets of antiviral host factors. Nevertheless, little is known about the silencing machinery in the nucleus that acts to prevent transcription or retroviral integration of extrachromosomal DNA. We here identified CHAF1A and CHAF1B as two players that mediate silencing of unintegrated HIV-1 DNAs. Our findings provide evidence that these factors act independently of their canonical nucleosome assembly complex to induce silencing early in infection. The characterization of the intrinsic cellular defense mechanism against incoming DNA is relevant to transient gene delivery, as mediated by virus-based vectors in gene therapy., Early events of the retroviral life cycle are the targets of many host restriction factors that have evolved to prevent establishment of infection. Incoming retroviral DNAs are transcriptionally silenced before integration in most cell types, and efficient viral gene expression occurs only after formation of the provirus. The molecular machinery for silencing unintegrated retroviral DNAs of HIV-1 remains poorly characterized. Here, we identified the histone chaperones CHAF1A and CHAF1B as essential factors for silencing of unintegrated HIV-1 DNAs. Using RNAi-mediated knockdown (KD) of multiple histone chaperones, we found that KD of CHAF1A or CHAF1B resulted in a pronounced increase in expression of incoming viral DNAs. The function of these two proteins in silencing was independent of their interaction partner RBBP4. Viral DNA levels accumulated to significantly higher levels in CHAF1A KD cells over controls, suggesting enhanced stabilization of actively transcribed DNAs. Chromatin immunoprecipitation assays revealed no major changes in histone loading onto viral DNAs in the absence of CHAF1A, but levels of the H3K9 trimethylation silencing mark were reduced. KD of the H3K9me3-binding protein HP1γ accelerated the expression of unintegrated HIV-1 DNAs. While CHAF1A was critical for silencing HIV-1 DNAs, it showed no role in silencing of unintegrated retroviral DNAs of mouse leukemia virus. Our study identifies CHAF1A and CHAF1B as factors involved specifically in silencing of HIV-1 DNAs early in infection. The results suggest that these factors act by noncanonical pathways, distinct from their histone loading activities, to mediate silencing of newly synthesized HIV-1 DNAs.

Moloney Murine Leukemia Virus (M-MLV) proviral DNA is transcriptionally silenced in embryonic cells by a large repressor complex tethered to the provirus by two sequence-specific DNA binding proteins, ZFP809 and YY1. A central component of the complex is Trim28, a scaffold protein that regulates many target genes involved in cell cycle progression, DNA damage responses, and viral gene expression. The silencing activity of Trim28, and its interactions with corepressors are often regulated by post-translational modifications such as sumoylation and phosphorylation. We defined the interaction domains of Trim28 and YY1, and investigated the role of sumoylation and phosphorylation of Trim28 in mediating M-MLV silencing. The RBCC domain of Trim28 was sufficient for interaction with YY1, and acidic region 1 and zinc fingers of YY1 were necessary and sufficient for its interaction with Trim28. Additionally, we found that residue K779 was critical for Trim28-mediated silencing of M-MLV in embryonic cells.

// Oded Danziger 1, * , Ben Shai 1, * , Yosef Sabo 1 , Eran Bacharach 1 , Marcelo Ehrlich 1 1 Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel * These authors contributed equally to this work Correspondence to: Marcelo Ehrlich, email: marceloe@post.tau.ac.il Eran Bacharach, email: eranba@tauex.tau.ac.il Keywords: human metapneumovirus, epizootic hemorrhagic disease virus, epigenetic silencing, interferon, prostate cancer cells Received: March 21, 2016 Accepted: May 13, 2016 Published: June 28, 2016 ABSTRACT Interferons (IFNs) induce anti-viral programs, regulate immune responses, and exert anti-proliferative effects. To escape anti-tumorigenic effects of IFNs, malignant cells attenuate JAK/STAT signaling and expression of IFN stimulated genes (ISGs). Such attenuation may enhance the susceptibility of tumor cells to oncolytic virotherapy. Here we studied genetic and epigenetic mechanisms of interference with JAK/STAT signaling and their contribution to susceptibility of prostate cancer cells to viral infection. Bioinformatics analysis of gene-expression in cohorts of prostate cancer patients revealed genetic and epigenetic interference with the IFN program. To correlate lack of IFN signaling and susceptibility to viral infection and oncolysis; we employed LNCaP prostate cancer cells as cellular model, and the human metapneumovirus and the epizootic hemorrhagic disease virus as infectious agents. In LNCaP cells, JAK1 is silenced by bi-allelic inactivating mutations and epigenetic silencing, which also silences ISGs. Chemical inhibition of epigenetic silencing partially restored IFN-sensitivity, induced low levels of expression of selected ISGs and attenuated, but failed to block, viral infection and oncolysis. Since viral infection was not blocked by epigenetic modifiers, and these compounds may independently-induce anti-tumor effects, we propose that epigenetic modifiers and virotherapy are compatible in treatment of prostate tumors defective in JAK1 expression and IFN signaling.

SummaryStable microtubule (MT) subsets form distinct networks from dynamic MTs and acquire distinguishing posttranslational modifications, notably detyrosination and acetylation. Acting as specialized tracks for vesicle and macromolecular transport, their formation is regulated by the end-binding protein EB1, which recruits proteins that stabilize MTs. We show that HIV-1 induces the formation of acetylated and detyrosinated stable MTs early in infection. Although the MT depolymerizing agent nocodazole affected dynamic MTs, HIV-1 particles localized to nocodazole-resistant stable MTs, and infection was minimally affected. EB1 depletion or expression of an EB1 carboxy-terminal fragment that acts as a dominant-negative inhibitor of MT stabilization prevented HIV-1-induced stable MT formation and suppressed early viral infection. Furthermore, we show that the HIV-1 matrix protein targets the EB1-binding protein Kif4 to induce MT stabilization. Our findings illustrate how specialized MT-binding proteins mediate MT stabilization by HIV-1 and the importance of stable MT subsets in viral infection.

N-myristoylation is the covalent attachment of myristic acid to the N terminus of proteins in eukaryotic cells. The matrix domain (MA) of HIV-1 Gag protein is N-myristoylated and plays an important role in virus budding. In screening for host factors that interact with HIV-1 MA, we found that heme oxygenase (HO-2) specifically binds the myristate moiety of Gag. HO-2 was also found to bind TRAM, an adaptor protein for Toll-like receptor 4 (TLR4), and thereby impact both virus replication and cellular inflammatory responses. A crystal structure revealed that HO-2 binds myristate via a hydrophobic channel adjacent to the heme-binding pocket. Inhibiting HO-2 expression, or blocking myristate binding with a heme analog, led to marked increases in virus production. HO-2 deficiency caused hyperresponsive TRAM-dependent TLR4 signaling and hypersensitivity to the TLR4 ligand lipopolysaccharide. Thus, HO-2 is a cellular myristate-binding protein that negatively regulates both virus replication and host inflammatory responses.

YXXL motifs in cellular and viral proteins have a variety of functions. The matrix (M) protein of the respiratory pathogen human metapneumovirus (hMPV) contains two such conserved motifs—YSKL and YAGL. We mutated these sequences to analyze their contributions to hMPV infectivity. The mutant clones were capable of intracellular replication; however, the YAGL but not YSKL mutants were defective at spreading in infected cultures. We improved the reverse genetics system for hMPV and generated cell lines that stably expressed selectable, replicating full-length genomes for both the wild type and the mutant clones, allowing microscopic and biochemical analyses of these viruses. YAGL mutants produced normal cellular levels of M protein but failed to release virions, while ectopic coexpression of wild-type M generated particles that were restricted to a single cycle of infection. The YAGL motif did not act as a late (L) domain, however, since hMPV budding was independent of the cellular endosomal sorting complex required for transport (ESCRT) machinery and because replacement of the YAGL motif with classical L domains generated defective viruses. Instead, the YAGL mutants had defective M assemblies lacking a normal filamentous appearance and showed poor extractability from the cell compared to the wild-type protein. The mutant proteins were not grossly misfolded, however, as they interacted with cellular membranes and coassembled with wild-type M proteins. Thus, the YAGL motif is an important determinant of hMPV assembly. Furthermore, the selectable hMPV genomes described here should extend the use of reverse genetics systems in the analysis of spreading-defective viruses.

Moloney murine leukemia virus (MMuLV) Gag protein contains three identified late (L) domains, PPPY, YPAL, and PSAP, which are thought to interact with the endosomal sorting machinery to assist budding. We created single and combined L-domain mutants in all permutations and tested the resulting clones for budding and replication. Budding and replication of all viruses with mutated PPPY were greatly reduced; however, the basal replication level was retained, demonstrated by the slow spread of the viruses in culture. Mutations in PSAP or YPAL did not affect budding or spreading, demonstrating that these two motifs are dispensable for efficient MMuLV replication. Furthermore, the basal budding level was maintained following inhibition of endosomal sorting machinery, emphasizing that the basal budding of MMuLV is independent of this machinery.

Background: Synonymous or silent mutations are usually thought to evolve neutrally. However, accumulating recent evidence has demonstrated that silent mutations may destabilize RNA structures or disrupt cis regulatory motifs superimposed on coding sequences. Such observations suggest the existence of stretches of codon sites that are evolutionary conserved at both DNA-RNA and protein levels. Such stretches may point to functionally important regions within protein coding sequences not necessarily reflecting functional constraints on the amino-acid sequence. The HIV-1 genome is highly compact, and often harbors overlapping functional elements at the protein, RNA, and DNA levels. This superimposition of functions leads to complex selective forces acting on all levels of the genome and proteome. Considering the constraints on HIV-1 to maintain such a highly compact genome, we hypothesized that stretches of synonymous conservation would be common within its genome. Results: We used a combined computational-experimental approach to detect and characterize regions exhibiting strong purifying selection against synonymous substitutions along the HIV-1 genome. Our methodology is based on advanced probabilistic evolutionary models that explicitly account for synonymous rate variation among sites and rate dependencies among adjacent sites. These models are combined with a randomization procedure to automatically identify the most statistically significant regions of conserved synonymous sites along the genome. Using this procedure we identified 21 conserved regions. Twelve of these are mapped to regions within overlapping genes, seven correlate with known functional elements, while the functions of the remaining four are yet unknown. Among these four regions, we chose the one that deviates most from synonymous rate homogeneity for in-depth computational and experimental characterization. In our assays aiming to quantify viral fitness in both early and late stages of the replication cycle, no differences were observed between the mutated and the wild type virus following the introduction of synonymous mutations. Conclusions: The contradiction between the inferred purifying selective forces and the lack of effect of these mutations on viral replication may be explained by the fact that the phenotype was measured in single-cycle infection assays in cell culture. Such a system does not account for the complexity of HIV-1 infections in vivo, which involves multiple infection cycles and interaction with the host immune system.