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Bacteria and phages are locked in a co-evolutionary arms race where each entity evolves mechanisms to restrict the proliferation of the other. Phage-encoded defense inhibitors have proven powerful tools to interrogate how defense systems function. A relatively common defense system is BREX (bacteriophage exclusion); however, how BREX functions to restrict phage infection remains poorly understood. A BREX system encoded by the sulfamethoxazole and trimethoprim (SXT) integrative and conjugative element, VchInd5, was recently identified in Vibrio cholerae, the causative agent of the diarrheal disease cholera. The lytic phage ICP1 (International Centre for Diarrhoeal Disease Research, Bangladesh cholera phage 1) that co-circulates with V. cholerae encodes the BREX-inhibitor OrbA, but how OrbA inhibits BREX is unclear. Here, we determine that OrbA inhibits BREX using a unique mechanism from known BREX inhibitors by directly binding to the BREX component BrxC. BrxC has a functional ATPase domain that, when mutated, not only disrupts BrxC function but also alters how BrxC multimerizes. Furthermore, we find that OrbA binding disrupts BrxC-BrxC interactions. We determine that OrbA cannot bind BrxC encoded by the distantly related BREX system encoded by the aSXT VchBan9, and thus fails to inhibit this BREX system that also circulates in epidemic V. cholerae. Lastly, we find that homologs of the VchInd5 BrxC are more diverse than the homologs of the VchBan9 BrxC. These data provide new insight into the function of the BrxC ATPase and highlight how phage-encoded inhibitors can disrupt phage defense systems using different mechanisms.IMPORTANCEWith renewed interest in phage therapy to combat antibiotic-resistant pathogens, understanding the mechanisms bacteria use to defend themselves against phages and the counter-strategies phages evolve to inhibit defenses is paramount. Bacteriophage exclusion (BREX) is a common defense system with few known inhibitors. Here, we probe how the vibriophage-encoded inhibitor OrbA inhibits the BREX system of Vibrio cholerae, the causative agent of the diarrheal disease cholera. By interrogating OrbA function, we have begun to understand the importance and function of a BREX component. Our results demonstrate the importance of identifying inhibitors against defense systems, as they are powerful tools for dissecting defense activity and can inform strategies to increase the efficacy of some phage therapies.

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. The expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix αG was resistant to wild-type VACV infection and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.IMPORTANCEThe molecular mechanisms that govern the host range of viruses are incompletely understood. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. The current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters double-stranded (ds) RNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete and that dsRNA sequestration is not the primary mechanism for E3 activity.

Hemipteran insects transmit viruses when infesting plants, during which vectors activate salicylic acid (SA)-regulated antiviral defenses. How vector-borne plant viruses circumvent these antiviral defenses is largely unexplored. During co-infections of begomoviruses and betasatellites in plants, betasatellite-encoded βC1 proteins interfere with SA signaling and reduce the activation of antiviral resistance. βC1 inhibits SA-induced degradation of NbNPR3 (Nicotiana benthamiana nonexpressor of pathogenesis-related genes 3), a negative regulator of SA signaling. βC1 does not bind directly to NbNPR3, but regulates NbNPR3 degradation via heat shock protein 90s (NbHSP90s). NbHSP90s bind to both NbNPR3 and βC1 and suppress SA signaling. This viral success strategy appears to be conserved as it is also documented for viral proteins encoded by two aphid-borne viruses. Our findings reveal an exquisite mechanism that facilitates the persistence of vector-borne plant viruses and provide important insights into the intricacies of the virus life cycle.

CD45 is a cell surface phosphatase that shapes the T cell receptor signaling threshold but does not have a known ligand. A family of adenovirus proteins, including E3/49K, exploits CD45 to evade immunity by binding to the extracellular domain of CD45, resulting in the suppression of T cell signaling. We determined the cryo-EM structure of this complex and found that the E3/49K protein is composed of three immunoglobulin domains assembled as beads on a string that compel CD45 into a closely abutted dimer by cross-linking the CD45 D3 domain, leading to steric inhibition of its intracellular phosphatase activity. Inspired by the E3/49K mechanism, we engineered CD45 surrogate ligands that can fine-tune T cell activation by dimerizing CD45 into different orientations and proximities. The adenovirus E3/49K protein has taught us that, despite a lack of a known ligand, CD45 activity can be modulated by extracellular dimerizing ligands that perturb its phosphatase activity and alter T cell responses.

ICP1, a lytic bacteriophage of Vibrio cholerae, is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs dependence on ICP1s DNA replication machinery and virion components results in inhibition of ICP1s lifecycle. PLEs are expected to depend on ICP1 factors for genome packaging, but the mechanism(s) PLEs use to inhibit ICP1 genome packaging is currently unknown. Here, we identify and characterize Gpi, PLEs indiscriminate genome packaging inhibitor. Gpi binds to ICP1s large terminase (TerL), the packaging motor, and blocks genome packaging. To overcome Gpis negative effect on TerL, a component PLE also requires, PLE uses two genome packaging specifiers, GpsA and GpsB, that specifically allow packaging of PLE genomes. Surprisingly, PLE also uses mimicry of ICP1s pac site as a backup strategy to ensure genome packaging. PLEs pac site mimicry, however, is only sufficient if PLE can inhibit ICP1 at other stages of its lifecycle, suggesting an advantage to maintaining Gpi, GpsA and GpsB. Collectively, these results provide mechanistic insights into another stage of ICP1s lifecycle that is inhibited by PLE, which is currently the most inhibitory of the documented phage satellites. More broadly, Gpi represents the first satellite-encoded inhibitor of a phage TerL.

The rapid evolution of viruses generates proteins that are essential for infectivity and replication but with unknown functions, due to extreme sequence divergence1. Here, using a database of 67,715 newly predicted protein structures from 4,463 eukaryotic viral species, we found that 62% of viral proteins are structurally distinct and lack homologues in the AlphaFold database2,3. Among the remaining 38% of viral proteins, many have non-viral structural analogues that revealed surprising similarities between human pathogens and their eukaryotic hosts. Structural comparisons suggested putative functions for up to 25% of unannotated viral proteins, including those with roles in the evasion of innate immunity. In particular, RNA ligase T-like phosphodiesterases were found to resemble phage-encoded proteins that hydrolyse the host immune-activating cyclic dinucleotides 3',3'- and 2',3'-cyclic GMP-AMP (cGAMP). Experimental analysis showed that RNA ligase T homologues encoded by avian poxviruses similarly hydrolyse cGAMP, showing that RNA ligase T-mediated targeting of cGAMP is an evolutionarily conserved mechanism of immune evasion that is present in both bacteriophage and eukaryotic viruses. Together, the viral protein structural database and analyses presented here afford new opportunities to identify mechanisms of virus-host interactions that are common across the virome.

The poxvirus-derived protein vCCI (viral CC chemokine inhibitor) binds almost all members of the CC chemokine family with nanomolar affinity, inhibiting their pro-inflammatory actions. Understanding the affinity and specificity of vCCI could lead to new anti-inflammatory therapeutics. CCL17, also known as TARC, is unusual among CC chemokines by having only micromolar binding to vCCI. We have used sequence analysis and molecular simulations to determine the cause of this weak binding, which identified several locations in CCL17 where mutations seemed likely to improve binding to vCCI. Based on the aforementioned analysis, we expressed and tested multiple mutants of CCL17. We found two single point mutants V44K and Q45R that increased binding affinity to vCCI by 2-3-fold and, in combination, further improved affinity by 7-fold. The CCL17 triple mutant G17R/V44K/Q45R yielded a Kd of 0.25 ± 0.13 μM, a 68-fold improvement in affinity compared to the complex with wild-type CCL17. A quadruple mutant G17R/V44K/Q45R/R57W showed high affinity (0.59 ± 0.09 μM) compared to the wild type but lower affinity than the triple mutant. This work demonstrates that sequence comparisons and molecular simulations can predict chemokine mutations that increase the level of binding to vCCI, an important first step in developing engineered chemokine inhibitors useful for anti-inflammatory therapy.

Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2-deoxyguanosine) are major products of 2-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer-dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to 2-deoxyguanosine. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G, or U. Fitting of reaction data indicates that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast- or slow-reacting base pairs vary less than twofold as a function of the nucleotide opposite the lesion. Promoter-dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side by side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

A new emissive guanosine analog CF3thG, constructed by a single trifluoromethylation step from the previously reported thG, displays red-shifted absorption and emission spectra compared to its precursor. The impact of solvent type and polarity on the photophysical properties of CF3thG suggests that the electronic effects of the trifluoromethyl group dominate its behavior and demonstrates its susceptibility to microenvironmental polarity changes. In vitro transcription initiations using T7 RNA polymerase, initiated with CF3thG, result in highly emissive 5'-labeled RNA transcripts, demonstrating the tolerance of the enzyme toward the analog. Viability assays with HEK293T cells displayed no detrimental effects at tested concentrations, indicating the safety of the analog for cellular applications. Live cell imaging of the free emissive guanosine analog using confocal microscopy was facilitated by its red-shifted absorption and emission and adequate brightness. Real-time live cell imaging demonstrated the release of the guanosine analog from HEK293T cells at concentration-gradient conditions, which was suppressed by the addition of guanosine.

Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the Nucleocytoviricota phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.

Influenza is one of the most prevalent viruses that has plagued millions worldwide. Every year health organizations encourage the public to get their flu vaccines to combat the flu season. Although the flu vaccines and medicines are widely available, it is estimated that flu viruses still cause 20,000-50,000 deaths every year. The two most common influenza virus strains are influenza A and B viruses, or IAV and IBV, respectively. It has been discovered that drug resistance develops very soon after a new drug is launched. It is highly demanded that anti-flu virus drugs with novel mechanisms be developed. Our lab has discovered that SUMOylation, a post-translational modification, is essential to the viral IAV and IBV life cycle. In this study, we have screened all the E3 ligases in the human genome to discover the SUMO E3 ligase responsible for the essential SUMOylation of IAV M1 protein using our Quantitative Fluorescence Energy Transfer(qFRET). We first determined the FRET spectrum of all E3 ligases with M1 protein and then quantified the FRET signals to provide a first-line examination of interactions. We then determined the E3-M1 interaction affinities, KD, to ensure the real interactions. We found the E3 ligase PIAS1 has the highest affinity to M1 among other E3s. By understanding the interaction affinity between IAV M1 protein with SUMOylation E3 ligase, we hope to block the interaction between the PIAS1-M1 for novel anti-flu medicine development.

Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

Phage viruses shape the evolution and virulence of their bacterial hosts. The Salmonella enterica genome encodes several stress-inducible prophages. The Gifsy-1 prophage terminase protein, whose canonical function is to process phage DNA for packaging in the virus head, unexpectedly acts as a transfer ribonuclease (tRNase) under oxidative stress, cleaving the anticodon loop of tRNALeu. The ensuing RNA fragmentation compromises bacterial translation, intracellular survival, and recovery from oxidative stress in the vertebrate host. S. enterica adapts to this transfer RNA (tRNA) fragmentation by transcribing the RNA repair Rtc system. The counterintuitive translational arrest provided by tRNA cleavage may subvert prophage mobilization and give the host an opportunity for repair as a way of maintaining bacterial genome integrity and ultimately survival in animals.

Nanoparticle-based antigen carrier systems have become a significant area of research with the advancement of nanotechnology. Biodegradable polymers have emerged as particularly promising carrier vehicles due to their ability to address the limitations of existing vaccine systems. In this study, we successfully encapsulated the G5-24 linear peptide, located between amino acids 253 and 275 in the primary sequence of the rabies virus G protein, into biodegradable and biocompatible PLGA copolymer using the double emulsion solvent evaporation method. The resulting nanoparticles had a size of approximately 230.9 ± 0.9074 nm, with a PDI value of 0.168 ± 0.017 and a zeta potential value of −9.86 ± 0.132 mV. SEM images confirmed that the synthesized nanoparticles were uniform in size and distribution. Additionally, FTIR spectra indicated successful peptide loading into the nanoparticles. The encapsulation efficiency of the peptide-loaded nanoparticles was 73.3%, with a peptide loading capacity of 48.2% and a reaction yield of 30.4%. Peptide release studies demonstrated that 65.55% of the peptide was released in a controlled manner over 28 d, following a 'biphasic burst release' profile consistent with the degradation profile of PLGA. This controlled release is particularly beneficial for vaccine studies. Cytotoxicity tests revealed that the R-NP formulation did not induce cytotoxicity in fibroblast cells and enhanced NO production in macrophages, indicating its potential for vaccine development. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) are essential components of innate immunity, maintaining the functionality of immune systems that control virus infection. However, how lncRNAs engage immune responses during influenza A virus (IAV) infection remains unclear. Here, we show that lncRNA USP30-AS1 is up-regulated by infection of multiple different IAV subtypes and is required for tuning inflammatory and antiviral response in IAV infection. Genetically inactivation of USP30-AS1 enhances viral protein synthesis and viral growth. USP30-AS1 is an interferon-stimulated gene, and the induction of USP30-AS1 can be achieved by JAK-STAT mediated signaling activation. The immune regulation of USP30-AS1 is independent of its proximal protein-coding gene USP30. In IAV infection, deletion of USP30-AS1 unleashes high systemic inflammatory responses involving a broad range of pro-inflammatory factors, suggesting USP30-AS1 as a critical modulator of immune responses in IAV infection. Furthermore, we established a database providing well-annotated host gene expression profiles IAV infection or immune stimulation. Author summary: Influenza A virus (IAV) infection can induce differential expression of long non-coding RNAs (lncRNAs). However, the understanding of IAV induced lncRNAs that involve in host immune responses is limited. Here we identified that lncRNA USP30-AS1 was induced by multiple subtypes of IAV infection, serving as a critical regulator regarding IAV induced immune responses. Deletion of USP30-AS1 led to enhanced viral protein synthesis and elevated viral growth in IAV infection. JAK-STAT signaling activation can drive the transcription of USP30-AS1. USP30-AS1 does not exert function through the nearby partner protein-coding gene USP30. Deficiency of USP30-AS1 triggers unbalanced, elevated pro-inflammatory responses in IAV infected cells, indicating the role of USP30-AS1 as a modulator regarding immune response during IAV infection. We also provide a user-friendly database that allows access to well-annotated host gene expression profiles in influenza virus infection or interferon stimulation. [ABSTRACT FROM AUTHOR]

Background Methods Results Conclusions Crimean‐Congo hemorrhagic fever is a tick‐borne zoonotic disease that may be severe and is present in many African countries. We aimed to understand the seroprevalence and risk for Crimean‐Congo hemorrhagic fever virus in Tanzania by testing archived serum samples from patients enrolled in a prospective cohort study.We prospectively enrolled febrile inpatients and outpatients from 2012 through 2014 at two referral hospitals in northern Tanzania. Archived serum samples were tested for Crimean‐Congo hemorrhagic fever virus antibodies initially by a Luminex assay screen followed by confirmation with immunofluorescence assay. Evidence of exposure to Crimean‐Congo hemorrhagic fever virus was defined as antibody detection by Luminex and confirmed by immunofluorescence assay. Questionnaire data were used to construct logistic regression models to understand factors associated with prior exposure to Crimean‐Congo hemorrhagic fever virus. Domains of predictor variables included sociodemographics, livestock‐rearing activities, and environmental factors.Of 735 participants included, antibodies to Crimean‐Congo hemorrhagic fever virus nucleocapsid protein were detected by Luminex assay in 23 (3.1%) and confirmed by immunofluorescence assay in 13 (1.8%). In multivariable logistic regression, prior exposure to Crimean‐Congo hemorrhagic fever virus was associated with self‐report of milking livestock in the past month (adjusted OR [aOR]: 12.6, 95% CI 1.6–99.8) and natural log increase in goat density (head/km2; aOR: 1.7, 95% CI: 1.1–2.7).We show serologic evidence of prior exposure to Crimean‐Congo hemorrhagic fever virus among humans in northern Tanzania. Similar to other settings, our results suggest that exposure is closely linked to livestock activities. Additional research is warranted to understand reservoirs and modes of transmission of Crimean‐Congo hemorrhagic fever virus to humans in northern Tanzania. [ABSTRACT FROM AUTHOR]

TLR4 is a single-pass transmembrane protein whose structure and function as a pattern recognition receptor (PRR) are highly conserved in mammals. TLR4 can be located on the cell surface and/or within cytoplasmic vesicles. TLR4 participates in direct and indirect interactions with viruses and viral proteins to trigger antiviral responses. In several instances, TLR4 polymorphisms can influence virulence. TLR4 agonists are being used to improve vaccine efficacy. Extracellular proteins, including antimicrobial peptides and danger-associated molecular patterns (DAMPs) impact TLR-mediated antiviral responses. Since the initial description of Toll receptors in Drosophila and their mammalian counterparts Toll-like receptors (TLRs), numerous fundamental and applied studies have explored their crucial role as sensors of pathogen-associated molecular patterns (PAMPs). Among the ten human TLRs, TLR4 is particularly well known for its ability to detect lipopolysaccharides (LPS), a component of the Gram-negative bacterial cell wall. In addition to its archetypal functions, TLR4 is also a versatile virus sensor. This review provides a background on the discovery of TLR4 and how this knowledge laid a foundation for characterization of its diverse roles in antiviral responses, examined through genetic, biochemical, structural, and immunological approaches. These advances have led to a deeper understanding of the molecular functions that enable TLR4 to orchestrate multi-nodal control by professional antigen-presenting cells (APCs) to initiate appropriate and regulated antiviral immune responses. [ABSTRACT FROM AUTHOR]

The receptor‐binding domain (RBD) of the SARS‐CoV‐2 virus spike protein has emerged as a promising target for the generation of neutralizing antibodies. Although the RBD subunit is more stable than its encoding mRNA, RBD is poorly immunogenic. It is hypothesized that this limitation can be overcome by sustained coadministration with a more potent and optimized adjuvant than standard adjuvants. One such candidate adjuvant, cGAMP, exhibits promising potency via activation of the antiviral STING pathway. Unfortunately, delivery of cGAMP as a therapeutic exhibits poor performance due to poor pharmacokinetics and pharmacodynamics from rapid excretion and degradation. To overcome these limitations, it is sought to create an artificial immunological niche enabling the slow release of cGAMP and RBD to mimic natural infections in which immune‐activating molecules are colocalized with antigen. Specifically, through coencapsulation of cGAMP and RBD in an injectable polymer‐nanoparticle (PNP) hydrogel, the cGAMP‐adjuvanted hydrogel vaccine elicits more potent, durable, and broad antibody responses with improved neutralization as compared to dose‐matched bolus controls and hydrogel‐based vaccines lacking cGAMP. The cGAMP‐adjuvanted hydrogel platform can be further explored for the delivery of other antigens to enhance immunity against a broad range of pathogens. [ABSTRACT FROM AUTHOR]

Vaccines are the most effective tool against COVID‐19 and are generally safe. Very rare and heterogeneous cases of acute liver injury associated to all types of SARS‐CoV‐2 vaccines have been reported, mostly with autoimmune features. Epidemiological studies used heterogeneous diagnostic criteria and included different populations. Immunological studies in selected cases of acute liver injury linked to mRNA SARS‐CoV‐2 vaccines suggest that it has a unique pathophysiology, the vaccine‐encoded spike protein playing a central role in triggering the aberrant immune response. In most series, liver injury was observed more often following the second vaccine dose. Latency from vaccination to the diagnosis of hepatitis was 1–147 days after the last vaccine dose. Raised immunoglobulin G levels and positive anti‐nuclear and/or anti‐smooth muscle antibodies are frequent. The vast majority of reported cases have been treated with corticosteroids, mostly associated with azathioprine. Outcome is generally favourable, but cases requiring liver transplantation or causing death have been reported. The heterogeneous clinical entity of acute liver injury linked to SARS‐CoV‐2 vaccines includes patients requiring long‐term immunosuppression, similarly to autoimmune hepatitis, and patients with self‐limiting liver damage, possibly representing a unique form of autoimmune‐like hepatitis, which we suggest being referred to as SARS‐CoV‐2 vaccine‐associated liver injury (SVALI). Further studies are needed to investigate the pathogenic mechanisms related to the immune response to the spike viral protein in the liver. [ABSTRACT FROM AUTHOR]

Deeper insight is needed on how monoclonal antibodies (mAbs) affect vaccine-mediated immune responses when targeting the same protein. We describe the first prospective randomised trial designed to understand mAb-mediated alterations in vaccine-induced immune responses to SARS-CoV-2 spike protein epitopes. This randomised, open-label, parallel-group study assessed the potential interaction of a mAb combination, casirivimab and imdevimab, with a vaccine, Moderna's mRNA-1273, in healthy SARS-CoV-2 immunologically naive, seronegative adults at six centres in the USA. Participants were randomly assigned (per prespecified randomisation ratios within enrolment waves) according to a computer-generated randomisation scheme, stratified by age (<65 years and ≥65 years), to various intravenous or subcutaneous doses of casirivimab and imdevimab before, after, or at the same time as mRNA-1273 or to mRNA-1273 only. The doses of casirivimab and imdevimab were chosen to mimic various time intervals between receipt of 1200 mg of the mAb and the first dose of a primary series with mRNA-1273. The primary endpoint was vaccine-induced 50% inhibitory dilution neutralising antibody titres to SARS-CoV-2 spike protein, 56 days after the first vaccination. Secondary endpoints included vaccine-induced total antibodies to SARS-CoV-2 antigens and incidence of treatment-emergent adverse events. Exploratory endpoints included blood-derived T-cell and B-cell responses. The per-protocol set was used for the analysis of the primary endpoint and included all randomly assigned participants who received both doses of the vaccine and completed the injection or infusion of casirivimab and imdevimab per protocol, had no evidence of SARS-CoV-2 infection in the past or in the 56 days after the first dose of vaccine, and did not receive any intervention outside of the study that could alter the immune response. Safety was assessed in the safety analysis set, which included all randomly assigned participants who had received one or more doses of mRNA-1273 or any study drug, and analysed based on treatment received. The study is registered with ClinicalTrials.gov , NCT04852978 , and is complete. Between April 29, 2021, and Nov 21, 2022, 807 participants were assessed for eligibility and 295 were randomly assigned. 293 participants were included in the safety analysis set and 260 were included in the per-protocol set. All vaccinated participants developed neutralising antibodies to SARS-CoV-2, with median titres above the published protective threshold (100 IU/mL) against the SARS-CoV-2 D614G variant (considered a reference strain at the time the initial COVID-19 vaccines were developed). Titres were decreased up to 4-fold (median titres 280–450 IU/mL for casirivimab and imdevimab vs 1160 IU/mL for vaccine only on day 56) when casirivimab and imdevimab was given 85 days or less before vaccination (150–1200 mg intravenously) or co-administered subcutaneously (600 mg or 1200 mg) with vaccination. Minimal reduction in neutralisation titres was observed in the 48 mg and 12 mg intravenous groups, corresponding to receipt of casirivimab and imdevimab 113 days and 169 days, respectively, before vaccination, and when administering the vaccine 6 days before the mAb. Across all groups, mAbs had a minimal effect on vaccine-induced total antibodies and T-cell responses to the spike protein. Casirivimab and imdevimab plus mRNA-1273 was generally well tolerated; a slight increase in treatment-emergent adverse events was observed in the casirivimab and imdevimab plus vaccine groups versus the vaccine-only group. Casirivimab and imdevimab administration before or at the time of COVID-19 vaccination reduced the elicitation of SARS-CoV-2 neutralising antibodies, but minimal effect was observed when vaccination occurred before mAb administration. Although the clinical significance of this decrease in neutralisation is unclear, this evidence suggests that further investigation of potential interactions could be warranted before concurrent clinical use of mAbs and vaccines targeting the same viral proteins as their main modes of action for the prevention or treatment of infectious diseases. Regeneron Pharmaceuticals and F Hoffmann-La Roche. [ABSTRACT FROM AUTHOR]

G protein‐coupled receptors (GPCRs) are pivotal in cellular signalling and drug targeting. Herpesviruses encode GPCRs (vGPCRs) to manipulate cellular signalling, thereby regulating various aspects of the virus life cycle, such as viral spreading and immune evasion. vGPCRs mimic host chemokine receptors, often with broader signalling and high constitutive activity. This review focuses on the recent advancements in structural knowledge about vGPCRs, with an emphasis on molecular mechanisms of action and ligand binding. The structures of US27 and US28 from human cytomegalovirus (HCMV) are compared to their closest human homologue, CX3CR1. Contrasting US27 and US28, the homotrimeric UL78 structure (HCMV) reveals more distance to chemokine receptors. Open reading frame 74 (ORF74; Kaposi's sarcoma‐associated herpesvirus) is compared to CXCRs, whereas BILF1 (Epstein–Barr virus) is discussed as a putative lipid receptor. Furthermore, the roles of vGPCRs in latency and lytic replication, reactivation, dissemination and immune evasion are reviewed, together with their potential as drug targets for virus infections and virus‐related diseases. [ABSTRACT FROM AUTHOR]

All viruses are obligate intracellular parasites that use host machinery to synthesize viral proteins. In infected eukaryotes, viral secreted and transmembrane proteins are synthesized at the endoplasmic reticulum (ER). Many viruses refashion ER membranes into bespoke factories where viral products accumulate while evading host pattern recognition receptors. ER processes are tightly regulated to maintain cellular homeostasis, so viruses must either conform to ER regulatory mechanisms or subvert them to ensure efficient viral replication. Reticulophagy is a catabolic process that directs lysosomal degradation of ER components. There is accumulating evidence that reticulophagy serves as a form of antiviral defense; we call this defense "xERophagy" to acknowledge its relationship to xenophagy, the catabolic degradation of microorganisms by macroautophagy/autophagy. In turn, viruses can subvert reticulophagy to suppress host antiviral responses and support efficient viral replication. Here, we review the evidence for functional interplay between viruses and the host reticulophagy machinery. Abbreviations: AMFR: autocrine motility factor receptor; ARF4: ADP-ribosylation factor 4; ARL6IP1: ADP-ribosylation factor-like 6 interacting protein 1; ATL3: atlastin GTPase 3; ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; BPIFB3: BPI fold containing family B, member 3; CALCOCO1: calcium binding and coiled coil domain 1; CAMK2B: calcium/calmodulin-dependent protein kinase II, beta; CANX: calnexin; CDV: canine distemper virus; CCPG1: cell cycle progression 1; CDK5RAP3/C53: CDK5 regulatory subunit associated protein 3; CIR: cargo-interacting region; CoV: coronavirus; CSNK2/CK2: casein kinase 2; CVB3: coxsackievirus B3; DAPK1: death associated protein kinase 1; DENV: dengue virus; DMV: double-membrane vesicles; EBOV: Ebola virus; EBV: Epstein-Barr Virus; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EMCV: encephalomyocarditis virus; EMV: extracellular microvesicle; ER: endoplasmic reticulum; ERAD: ER-associated degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signalling 1; EV: extracellular vesicle; EV71: enterovirus 71; FIR: RB1CC1/FIP200-interacting region; FMDV: foot-and-mouth disease virus; HCMV: human cytomegalovirus; HCV: hepatitis C virus; HMGB1: high mobility group box 1; HSPA5/BiP: heat shock protein 5; IFN: interferon; IFNG/IFN-γ: interferon gamma; KSHV: Kaposi's sarcoma-associated herpesvirus; LIR: MAP1LC3/LC3-interacting region; LNP: lunapark, ER junction formation factor; MAP1LC3: microtubule-associated protein 1 light chain 3; MAP3K5/ASK1: mitogen-activated protein kinase kinase kinase 5; MAPK/JNK: mitogen-activated protein kinase; MeV: measles virus; MHV: murine hepatitis virus; NS: non-structural; PDIA3: protein disulfide isomerase associated 3; PRR: pattern recognition receptor; PRRSV: porcine reproductive and respiratory syndrome virus; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RETREG1/FAM134B: reticulophagy regulator 1; RHD: reticulon homology domain; RTN3: reticulon 3; RTN3L: reticulon 3 long; sAIMs: shuffled Atg8-interacting motifs; SARS-CoV: severe acute respiratory syndrome coronavirus; SINV: Sindbis virus; STING1: stimulator of interferon response cGAMP interactor 1; SVV: Seneca Valley virus; SV40: simian virus 40; TEX264: testis expressed gene 264 ER-phagy receptor; TFEB: transcription factor EB; TRAF2: TNF receptor-associated factor 2; UIM: ubiquitin-interacting motif; UFM1: ubiquitin-fold modifier 1; UPR: unfolded protein response; VAPA: vesicle-associated membrane protein, associated protein A; VAPB: vesicle-associated membrane protein, associated protein B and C; VZV: varicella zoster virus; WNV: West Nile virus; XBP1: X-box binding protein 1; XBP1s: XBP1 spliced; xERophagy: xenophagy involving reticulophagy; ZIKV: Zika virus [ABSTRACT FROM AUTHOR]

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To prevent H9N2 avian influenza virus (AIV) and Avian metapneumonovirus/C (aMPV/C) infections, we constructed recombinant aMPV/C viruses expressing the HA protein of H9N2 AIV. In addition, EGFP was inserted into the intermediate non-coding region of P-M protein in the aMPV/C genome using a reverse genetic system. The conditions for rescuing the recombinant virus were enhanced followed by insertion of the H9N2 AIV HA gene into the same location in the aMPV/C. The constructed recombinant virus raMPV/C-HA expressed the H9N2 AIV HA protein and showed good stability. Immunization of chicks with raMPV/C-HA increased the generation of neutralizing antibodies against aMPV and H9N2 AIV for 21 days. In the late challenge experiment, raMPV/C-HA effectively inhibited the replication of the virus in vivo , decreased the incidence of infection and conferred protection effects. [ABSTRACT FROM AUTHOR]

Differentiation therapy with all-trans retinoic acid (ATRA) is well established for acute promyelocytic leukemia (APL). However, the narrow application and tolerance development of ATRA remain to be improved. A number of kinase inhibitors have been reported to induce cell differentiation. In this study, we investigated several combinations of these kinase inhibitors. Recently, we revealed that the Akt inhibitor triciribine (TCN) efficiently induces differentiation of NB4 APL cells and acute myeloid leukemia (AML) M2-derived HL-60 cells through activation of the ERK/MAPK pathway. In the present study, we found that the p38 MAPK inhibitor PD169316 had profoundly enhanced the TCN effect for differentiation of NB4 and HL-60 cells. Morphologically, the combination of these two agents efficiently reduced the nuclear-to-cytoplasmic ratio and induced the expression of myelomonocytic markers (CD11b, CD11c) and some ectopic markers (erythroid glycophorin A, lymphoid CD7 and CD20), as determined by PCR and flow cytometry analyses. Western blotting analysis revealed that these agents efficiently induced phosphorylation of ERK. To clarify the molecular mechanisms involved in the TCN and PD169316-induced differentiation, we performed microarray analyses using NB4 cells. Pathway analysis using DAVID software indicated that "viral protein interaction with cytokine and cytokine receptor" and "cytokine-cytokine receptor interaction" were enriched with high significance. Real-time PCR analysis demonstrated that genes for components of these pathways, including chemokines like CCL1, CCL2, CCL3, CCL5, and CXCL8 as well as cytokines and receptors like CSF1, IL-10, IL-10RA, IL-10RB, IL-1β, and TNFSF10, were upregulated in NB4 and HL-60 cells during TCN and PD169316-induced differentiation. [ABSTRACT FROM AUTHOR]

Background: A strong association between multiple sclerosis (MS) and Epstein-Barr virus (EBV) has been established but the exact role of EBV in MS remains controversial. Recently, molecular mimicry between EBNA1 and specific GlialCAM, CRYAB and ANO2 peptides has been suggested as a possible pathophysiological mechanism. The aim of this study was to analyse anti-EBV antibodies in MS patients against (I) EBV lifecycle proteins, (II) putative cross-reactive peptides, and (III) during treatment. Methods: In this retrospective cross-sectional study, 258 serum samples were included consisting of EBV-negative (n = 25) and EBV-positive (n = 36) controls, 192 MS samples including untreated relapsing-remitting MS (RRMS) with and without relapses, secondary progressive MS (SPMS) and primary progressive MS (PPMS) patients, and 106 patients on 8 different treatment regimens. IgG and IgM antibody titers against EBV docking/fusion proteins (gp350, gh/gp42, gh/gL/gp42), immediate early antigen (BZLF1), early antigens (EA p85, EA P138, EA P54), capsid antigens (VCA P18, VCA P23, VCA gp125) and late antigens (EBNA1) were measured. Specific EBNA1 and GlialCAM, CRYAB and ANO2 peptides were synthesized and also incorporated in our custom magnetic bead based multiplex assay. Results: We observed significantly elevated IgG antibody titers in EBV-positive controls, RRMS with and without relapse, SPMS and PPMS patients for all lifecycle antigens except for several early antigens when compared to EBV-negative controls. Significantly higher IgG antibody titers were observed in RRMS patients for fusion proteins and EBNA1 peptides when compared to EBV-positive controls. An MS specific response was observed for ANO2 but not for GlialCAM or CRYAB. No significant treatment effects or a specific IgM response were detectable. Conclusion: The MS-specific, differential antibody response to EBV antigens confirms an altered immunological response to EBV in MS patients. EBV reactivation does not appear to play an important role in MS pathogenesis and no differential antibody signatures were observed between MS disease phases. The MS-specific anti-ANO2 antibody response suggests a potential role for EBNA1 as an antigenic driver, although the exact role of anti-ANO2 antibodies needs to be determined. The precise pathophysiological role of EBV in MS remains uncertain and requires further investigation. [ABSTRACT FROM AUTHOR]

SARS-CoV-2, the cause of COVID-19, primarily targets lung tissue, leading to pneumonia and lung injury. The spike protein of this virus binds to the common receptor on susceptible tissues and cells called the angiotensin-converting enzyme-2 (ACE2) of the angiotensin (ANG) system. In this study, we produced chimeric Macrobrachium rosenbergii nodavirus virus-like particles, presenting a short peptide ligand (ACE2tp), based on angiotensin-II (ANG II), on their outer surfaces to allow them to specifically bind to ACE2-overexpressing cells called ACE2tp-MrNV-VLPs. Replacing the ACE2tp at the protruding domains (P-domain) of the MrNV capsid proteins did not affect their normal assembly into icosahedral VLPs. The presentation of the ACE2tp on the P-domains significantly improved the binding and internalization of ACE2tp-MrNV-VLPs to hACE2-overexpressing HEK293T cells in a concentration-dependent manner. Furthermore, ACE2tp-MrNV-VLPs exhibited the ability to block the binding and infection of SARS-CoV-2 pseudovirus variants, including Wuhan, BA.2 Omicron, and Delta subtypes. Our results suggest that chimeric ACE2tp-MrNV-VLPs can serve as a blocking agent against various SARS-CoV-2 mutated variants and could also potentially serve as target-specific nano-containers to carry therapeutic agents to combat SARS-CoV-2 infections in the future. [ABSTRACT FROM AUTHOR]

Plant viruses have evolved different viral suppressors of RNA silencing (VSRs) to counteract RNA silencing which is a small RNA-mediated sequence-specific RNA degradation mechanism. Previous studies have already shown that the coat protein (CP) of cucumber mosaic virus (CMV) reduced RNA silencing suppression (RSS) activity of the VSR of CMV, the 2b protein. To demonstrate the universality of this CP-VSR interference, our study included three different viruses: CMV and peanut stunt virus (PSV) from the Bromoviridae, and plum pox virus (PPV) from the Potyviridae family. The RSS activity of the three VSRs (CMV 2b, PSV 2b, and PPV HC-Pro) was compared using Agrobacterium-mediated transient expression in Nicotiana benthamiana and the effect of CMV CP, PSV CP and PPV CP was validated on the RSS activity of their cognate and heterologous VSRs as well. Furthermore, the VSRs were also evaluated in PTGS suppressor-deficient CMV mutant (CMV NVE/10–12/AAA) virus-infected plants. The joint presence of CPs and VSRs resulted in decreased RSS activity in each combination, regardless of the origin of the two proteins, suggesting a universal role of the viral CPs in fine tuning of RSS. Interestingly the PSV CP elicited the strongest negative effect on the RSS activity of all three VSRs. [ABSTRACT FROM AUTHOR]

Human ANP32A/B (huANP32A/B) poorly support the polymerase activity of avian influenza viruses (AIVs), thereby limiting interspecies transmission of AIVs from birds to humans. The SUMO-interacting motif (SIM) within NS2 promotes the adaptation of AIV polymerase to huANP32A/B via a yet undisclosed mechanism. Here we show that huANP32A/B are SUMOylated by the E3 SUMO ligase PIAS2α, and deSUMOylated by SENP1. SUMO modification of huANP32A/B results in the recruitment of NS2, thereby facilitating huANP32A/B-supported AIV polymerase activity. Such a SUMO-dependent recruitment of NS2 is mediated by its association with huANP32A/B via the SIM-SUMO interaction module, where K68/K153-SUMO in huANP32A or K68/K116-SUMO in huANP32B interacts with the NS2-SIM. The SIM-SUMO-mediated interactions between NS2 and huANP32A/B function to promote AIV polymerase activity by positively regulating AIV vRNP-huANP32A/B interactions and AIV vRNP assembly. Our study offers insights into the mechanism of NS2-SIM in facilitating AIVs adaptation to mammals. This study reveals how avian influenza viruses adapt to human hosts. It shows that viral infection upregulates human ANP32A/B SUMOylation, which recruits the viral NS2 protein, boosting polymerase activity and aiding early infection in human cells. [ABSTRACT FROM AUTHOR]

Nonstructural protein 3C, a master protease of Picornaviridae, plays a critical role in viral replication by directly cleaving the viral precursor polyprotein to form the viral capsid protein and antagonizing the host antiviral response. Additionally, 3C protease, as a tool enzyme, is involved in regulating polyprotein expression. Here, the 3C mutant gene (3Cm), fused with a small ubiquitin-like modifier (SUMO) tag at the N-terminal and featuring a mutation at position 127, was inserted into the cold-shock plasmid pCold of Escherichia coli for expression. Meanwhile, the P1-∆2A plasmid was constructed for expression in Pichia pastoris. The expressions of 3C protein and P1 precursor protein were confirmed by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (SDS-PAGE), and western blot (WB) analysis. The results showed that the wild-type 3C protease is toxic to the host, not only inhibiting protein expression but also inducing the degradation of the host. Moreover, mutation of the 127th amino acid from leucine (L) to proline (P) on the β-ribbon of 3C enhanced the overexpression capacity of 3C in E. coli while maintaining enzymatic activity. Subsequently, 100 µg P1 protein was utilized as a substrate to investigate the cleavage efficiency of 3C protease at various concentrations, temperatures, durations, and pH levels. The results showed that the target protein was cleaved when the protease reached 8 μg. We also found that the presence of the N-terminal SUMO tag did not affect the cleavage activity of 3Cm. The optimal cleavage activity was observed between 25 and 37 °C, with the peak cleavage efficiency of 89% at 30 °C for 2 h. More than 50% of the substrate was degraded within 1 h at 30 °C. Its optimal pH range is between 7 and 8. Remarkably, the P1 protein, cleaved by 3Cm protease, can further form virus-like particles (VLPs) in vitro. Key points: • Expression and purification of toxic protein 3C protease in E. coli • Cleavage efficiency assessment of 3C protease at various temperatures, durations, and pH • Assembly of virus-like particles of FMDV by cleaving the precursor polyprotein in vitro [ABSTRACT FROM AUTHOR]

Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of nonenveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoVs) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase-separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA. [ABSTRACT FROM AUTHOR]

Potato, like other Solanaceae plants, have a small family of eukaryotic translation initiation factors 4E (eIF4E), including four proteins eIF4E-1, eIF4E-2, eIF(iso)4E, and the novel cap-binding protein (nCBP). These factors play roles in the process of the post-transcriptional regulation of gene expression. The eIF4Es are also used by numerous RNA viruses to synthesize their proteins. The quantitative detection of eIF4Es at the proteome level yields data about their abundances and the involvement of each factor in the process of translation initiation under normal and stress conditions. To quantify eIF4Es, we have developed a mass spectrometry protocol for the detection of isoforms in potato S. tuberosum leaf tissues by multiple reaction monitoring (MRM) assay. As a result, we have detected all potato eIF4Es and determined their amounts with the most abundant protein, eIF4E-1. [ABSTRACT FROM AUTHOR]

COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) had an adverse effect globally because it caused a global pandemic with several million deaths. This virus possesses spike protein that is cleaved or activated by Furin-like protease enzymes occurring by mammalian lung or respiratory cells to enter the mammalian body. The addition of the Furin cleavage site in SARS-CoV-2 makes it a more infectious and emerging virus than its ancestor's viruses. Phylogenetic relationships of coronavirus spike proteins have analyzed and mapped Furin recognition motif on the tree using bioinformatics tools such as GTEx, KEGG, GO, NCBI, PolyPhen-2, SNAP2, PANTHER, Hidden Markov Models (Fathmm), Phd-single-nucleotide polymorphism (SNP), I-TASSER, Modpred, Phobius, SIFT, iPTREE-STAB, and PROVEAN. During this study, it has been found that in certain regions, Furin SNPs have some relation with the susceptibility to SARS-CoV-2. Whereas in other regions, the effects are very negligible. Finally, our study demonstrates that Furin SNPs have a strong relationship with susceptibility to SARS-CoV-2. As it helps to cleave the spike protein of the virus, thus it can be targeted to inhibit at a particular site to prevent the SARS-CoV-2 from the entrance into the body. [ABSTRACT FROM AUTHOR]

Following COVID-19 outbreak with its unprecedented effect on the entire world, the interest to the coronaviruses increased. The causative agent of the COVID-19, severe acute respiratory syndrome coronavirus – 2 (SARS-CoV-2) is one of seven coronaviruses that is pathogenic to humans. Others include SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E. The viruses differ in their pathogenicity. SARS-CoV, MERS-CoV, and SARS-CoV-2 are capable to spread rapidly and cause epidemic, while HCoV-HKU1, HCoV-OC43, HCoV-NL63 and HCoV-229E cause mild respiratory disease. The difference in the viral behavior is due to structural and functional differences. All seven human coronaviruses possess four structural proteins: spike, envelope, membrane, and nucleocapsid. Spike protein with its receptor binding domain is crucial for the entry to the host cell, where different receptors on the host cell are recruited by different viruses. Envelope protein plays important role in viral assembly, and following cellular entry, contributes to immune response. Membrane protein is an abundant viral protein, contributing to the assembly and pathogenicity of the virus. Nucleocapsid protein encompasses the viral RNA into ribonucleocapsid, playing important role in viral replication. The present review provides detailed summary of structural and functional characteristics of structural proteins from seven human coronaviruses, and could serve as a practical reference when pathogenic human coronaviruses are compared, and novel treatments are proposed. [ABSTRACT FROM AUTHOR]

MHC B2 haplotype chickens have been reported to induce strong immune response against various avian pathogens. However, little is known about the CD8+T-cell epitope with MHC B2-restricted on subgroup J avian leukosis virus (ALV-J). In this study, we explored the ALV-J-induced cellular immune response in B2 haplotype chickens in vivo. We found that ALV-J infection significantly increased the proportion of CD8+T cells in chickens and up-regulated the expression of cytotoxic genes like Granzyme A and antiviral genes like IFIT5 at 14 days post-infection (dpi). We selected 32 candidate peptides based on the peptide-binding motif and further identified three MHC B2-restricted CD8+T epitopes on ALV-J, including Pol652−660, Gag374−382, and Gag403−411 which induced significant levels of chicken IFN-γ production in splenocytes from ALV-J infected chickens using the ELISpot assay. In addition, we also verified that the three identified epitopes stimulated memory splenocytes elevating TNF-α and IL-2 protein expression. Importantly, we found that the three positive peptides were highly conserved among ALV-A, ALV-B, ALV-E, ALV-J, and ALV-K. Taken together, we identified three MHC B2-restricted CD8+T cell epitopes on ALV-J, providing a foundation for developing effective T cell epitope vaccines targeting conserved internal viral proteins. [ABSTRACT FROM AUTHOR]

Senecavirus A (SVA) is an emerging pathogen that causes idiopathic vesicular infections in pig herds, posing a potential threat to their production performance. Heat shock protein 70 (Hsp70) is a molecular chaperone that plays an important role in host homeostasis under both physiological and stress conditions. However, the effects of Hsp70 on SVA infection and its underlying regulatory mechanisms remain unclear. Here, we confirmed that Hsp70 expression promotes SVA infection, as evidenced by the expression of viral proteins, viral titers, and the number of rSVA-eGFP-infected cells. This positive regulatory role of Hsp70 is mainly involved in post-entry stages of SVA. Viral proteins that interacted with Hsp70 were screened, and co-immunoprecipitation (co-IP) shows an interaction between Hsp70 and SVA L and 3D proteins. Subsequently, we determined that the expression of Hsp70 is beneficial for the stability of the SVA L and 3D proteins. Additionally, the substrate-binding domain (SBD) of Hsp70 plays an important role in the interaction between Hsp70 and SVA L or 3D proteins; and the deletion of this domain results in the loss of the stabilizing effect of Hsp70 on SVA L and 3D proteins and the positive regulatory effect of Hsp70 on SVA replication. These results reveal that Hsp70 promotes SVA infection by stabilizing viral L and 3D proteins and provides a strategy for preventing and controlling SVA infection. [ABSTRACT FROM AUTHOR]

Currently, a large number of novel tick-borne viruses potentially pathogenic to humans are discovered. Studying many of them by classical methods of virology is difficult due to the absence of live viral particles or a sufficient amount of their genetic material. In this case, the use of modern methods of bioinformatics and synthetic and structural biology can help. Haseki tick virus (HSTV) is a recently discovered tick-borne unclassified ssRNA(+) virus. HSTV-positive patients experienced fever and an elevated temperature. However, at the moment, there is no information on the tertiary structure and functions of its proteins. In this work, we used AlphaFold 3 and other bioinformatic tools for the annotation of HSTV nonstructural proteins, based on the principle that the tertiary structure of a protein is inextricably linked with its molecular function. We were the first to obtain models of tertiary structures and describe the putative functions of HSTV nonstructural proteins (NS3 helicase, NS3 protease, NS5 RNA-dependent RNA-polymerase, and NS5 methyltransferase), which play a key role in viral genome replication. Our results may help in further taxonomic identification of HSTV and the development of direct-acting antiviral drugs, POC tests, and vaccines. [ABSTRACT FROM AUTHOR]

Background The advent of short-course, curative treatment with direct-acting antivirals (DAA) has given promise for the global elimination of hepatitis C virus (HCV) infections by 2030. Virological failure occurs in 2%–12% of persons receiving curative DAA treatment and may be presaged by pre-existing polymorphisms or result from selection of drug resistant variants during therapy. Methods We conducted a systematic review to assess the prevalence of HCV resistance associated substitutions (RAS) among individuals with chronic hepatitis C infection who had virological failure following initial or re-treatment with pan-genotypic DAA regimens. We included 34 and 22 studies assessing RAS in people with virological failure published between January 2014 and July 2023. Pooled RAS prevalence was estimated using random-effects meta-analysis. Results The pooled prevalence of RAS in people with virological failure following initial DAA treatment was 78.0% (95% confidence interval [CI]: 62.0–92.0) for sofosbuvir/velpatasvir, 81.0% (95% CI: 67.0–93.0) for sofosbuvir/daclatasvir, and 79.0% (95% CI: 70.0–87.0) for glecaprevir/pibrentasvir, with a high prevalence of resistance to the NS5A inhibitors. Among those with virological failure following re-treatment regimens, RAS were present in 93.0% (95% CI: 83.0–99.0) for sofosbuvir/velpatasvir/voxilepravir and in 100% (95% CI: 92.0–100) for glecaprevir/pibrentasvir, with resistance driven by RAS to NS5A inhibitors. Discussion At least 1 RAS is present in a high proportion of the few individuals with virological failure following initial or re-treatment with pan-genotypic DAA regimens. There is a need for ongoing surveillance for DAA-associated resistance, to assess risk factors for their development and clinical impact to inform best practice strategies for re-treatment. [ABSTRACT FROM AUTHOR]

Host cell-encoded deaminases act as antiviral restriction factors to impair viral replication and production through introducing mutations in the viral genome. We sought to understand whether deaminases are involved in SARS-CoV-2 mutation and replication, and how the viral factors interact with deaminases to trigger these processes. Here, we show that APOBEC and ADAR deaminases act as the driving forces for SARS-CoV-2 mutagenesis, thereby blocking viral infection and production. Mechanistically, SARS-CoV-2 nucleocapsid (N) protein, which is responsible for packaging viral genomic RNA, interacts with host deaminases and co-localizes with them at stress granules to facilitate viral RNA mutagenesis. N proteins from several coronaviruses interact with host deaminases at RNA granules in a manner dependent on its F17 residue, suggesting a conserved role in modulation of viral mutagenesis in other coronaviruses. Furthermore, mutant N protein bearing a F17A substitution cannot localize to deaminase-containing RNA granules and leads to reduced mutagenesis of viral RNA, providing support for its function in enhancing deaminase-dependent viral RNA editing. Our study thus provides further insight into virus-host cell interactions mediating SARS-CoV-2 evolution. Synopsis: Host cell-encoded deaminases act as innate restriction factors that enhance viral genome mutation and evolution. This study shows that SARS-CoV-2 nucleocapsid (N) protein interacts with host deaminases at RNA granules, thus promoting viral mutagenesis. N protein specifically interacts with host ADAR and APOBEC deaminases. N protein localises to deaminase-containing RNA granules via RNA-dependent phase separation. N protein promotes deaminase-induced RNA editing of SARS-CoV-2 genome. N protein with a F17A mutation fails to localise to deaminase-containing RNA granules and reduces host deaminase-dependent mutagenesis of SARS-CoV-2 genome. Coronaviral N protein interacts and co-condensates with host ADAR and APOBEC deaminases to promote viral RNA editing. [ABSTRACT FROM AUTHOR]

The article from PLoS Pathogens discusses the role of SUMOylation in plants, a posttranslational modification process that can influence protein function and interactions. The study highlights how SUMOylation is involved in plant defense responses against pathogens, such as bacteria, and how viruses can manipulate the SUMO machinery to promote their own replication and evade host defenses. The complex interplay between SUMOylation and viral infections in plants underscores the importance of understanding these interactions for advancements in plant virology and agricultural biotechnology. [Extracted from the article]

Background Dengue vascular permeability syndrome is the primary cause of death in severe dengue infections. The protective versus potentially pathogenic role of dengue nonstructural protein-1 (NS1) antibodies are not well understood. The main goal of this analysis was to characterize the relationship between free NS1 concentration and NS1 antibody titers in primary and secondary dengue infection to better understand the presence and duration of NS1 antibody complexes in clinical dengue infections. Methods Hospitalized participants with acute dengue infection were recruited from Northern Colombia between 2018 and 2020. Symptom assessment, including dengue signs and symptoms, chart review, and blood collection, was performed. Primary versus secondary dengue was assessed serologically. NS1 titers and anti-NS1 antibodies were measured daily. Results Patients with secondary infection had higher antibody titers than those in primary infection, and there was a negative correlation between anti-NS1 antibody titer and NS1 protein. We demonstrate that in a subset of secondary infection, there were indeed NS1 antigen-antibody complexes on the admission day during the febrile phase that were not detectable by the recovery phase. Furthermore, dengue infection status was associated with higher circulating sialidases. Discussion The negative correlation between antibody and protein suggests that antibodies may play a role in clearing this viral protein. [ABSTRACT FROM AUTHOR]

Influenza virus, adenovirus, and respiratory syncytial virus cause major respiratory infections. These infections have similar initial symptoms making it difficult to differentiate them based on symptoms alone. PCR is currently used as the standard diagnostic test for these infections, however, it has its limitations such as non-specific and false-negative amplifications, high cost, and the inability to distinguish between a live or dead virus. Therefore, there is a need for alternative diagnostic methods that focus on protein. Here, we introduce TN-cyclon™, which is an enzyme-linked immunosorbent assay combined with thio-nicotinamide adenine dinucleotide cycling to amplify signals, rather than the protein itself. Using this method, we were able to detect extremely low levels of viruses such as influenza A, influenza B, adenovirus, and RS virus, with LODs of 2.96 × 10-18 moles/assay, 2.98 × 10-18 moles/assay, 2.36 × 10-18 moles/assay, and 3.55 × 10-18 moles/assay, respectively. Furthermore, we successfully detected viruses diluted with extract buffer, with a significant difference to the blank at concentrations of 3 pfu/mL for influenza A, 1000 pfu/mL for influenza B, 43.8 pfu/mL for adenovirus, and 125 pfu/mL for RS virus. This shows that our low-cost and easy-to-use technique has sufficient sensitivity in diagnosing respiratory infections. [ABSTRACT FROM AUTHOR]

The capsid proteins of many viruses are capable of spontaneous self-assembly into virus-like particles (VLPs), which do not contain the viral genome and are therefore not infectious. VLPs are structurally similar to their parent viruses and are therefore effectively recognized by the immune system and can induce strong humoral and cellular immune responses. The structural features of VLPs make them an attractive platform for the development of potential vaccines and diagnostic tools. Chimeric VLPs can be obtained by attaching foreign peptides to capsid proteins. Chimeric VLPs present multiple copies of the antigen on their surface, thereby increasing the effectiveness of the immune response. Recombinant VLPs can be produced in different expression systems. Plants are promising biofactories for the production of recombinant proteins, including VLPs. The main advantages of plant expression systems are the overall low cost and safety of plant-produced products due to the absence of pathogens common to plants and animals. This review provides an overview of the VLP platform as an approach to developing plant-produced vaccines, focusing on the use of transient expression systems. [ABSTRACT FROM AUTHOR]

It has been four years since the emergence of the COVID-19 pandemic, and the ongoing threat it poses to human health and life underscores the continued need for the development of antiviral medications as a means of mitigating future viral outbreaks. In this study, we present a novel class of antiviral compounds, naphthoquinone-fused enediyne sugar polysulfates, which have demonstrated efficacy against coronaviruses by targeting the conserved receptor binding domain on spike proteins. These compounds induce irreversible damage to the viral proteins that are essential for binding to host cells, resulting in inhibition of viral infection at nanomolar concentrations with minimal cytotoxic effects. Notably, the selectivity index of these compounds exceeds 50 000, suggesting significant potential for further development in antiviral therapeutics against coronavirus. [ABSTRACT FROM AUTHOR]

Dengue poses a persistent and widespread threat with no effective antiviral drug available till now. Several inhibitors have been developed by targeting the viral non-structural proteins including methyl transferase (NS5) of the dengue virus with possible therapeutic values. In this work, virtual screening, molecular docking, molecular dynamics simulations (200 ns), and assessments of free energy changes have been carried out to identify potential candidates from a database of flavonoids (ca. 2000) that may have good curative potential from the disease. The binding affinity of flavonoids, namely Genistein-7-glucoside (FLD1), 6'–O-Acetylgenistin (FLD2), 5,6-dihydroxy-2-(4-hydroxyphenyl)-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxane-2-yl]oxychromen-4-one (FLD3), Glucoliquiritigenin (FLD4), and Chrysin-7-O-glucoronide (FLD5) showed the binding affinities of −10.2, −10.2, −10.1, −10.1, −9.9 kcal/mol, respectively, and possessed better values than that of the native ligand (−7.6 kcal/mol) and diclofenac sodium (−7.3 kcal/mol). Drug-likeness of the top five flavonoids were acceptable and no end-point toxicity was hinted by ADMET predictions. The stability of the protein-ligand complexes was accessed from 200 ns molecular dynamics simulations in terms of various geometrical parameters; RMSD, RMSF of residues, Rg, SASA, H-bond, and RPDF. The binding free energy changes of these adducts were calculated by the MM/PBSA solvation model with negative values (from −38.01±7.53 to −17.75±11.03 kcal/mol) indicating the sustained spontaneity of the forward reaction and favorability of the product formation. The geometrical and thermodynamic parameters inferred that the flavonoids could bind at the orthosteric site of the target protein of DENV-2 and could inhibit its functioning, possibly, resulting in the prevention of the disease. Overall, this study highlights the anti-DENV activity of five flavonoids, positioning them as promising candidates for further development as antiviral agents against dengue infection. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus (HPV) causes disease through complex interactions between viral and host proteins, with the PI3K signaling pathway playing a key role. Proteins like AKT, IQGAP1, and MMP16 are involved in HPV-related cancer development. Traditional methods for studying protein-protein interactions (PPIs) are labor-intensive and time-consuming. Computational models are becoming more popular as they are less labor-intensive and often more efficient. This study aimed to develop a deep learning model to predict interactions between HPV and host proteins. Method: To achieve this, available HPV and host protein interaction data was retrieved from the protocol of Eckhardt et al and used to train a Recurrent Neural Network algorithm. Training of the model was performed on the SPYDER (scientific python development environment) platform using python libraries; Scikit-learn, Pandas, NumPy, and TensorFlow. The data was split into training, validation, and testing sets in the ratio 7:1:2, respectively. After the training and validation, the model was then used to predict the possible interactions between HPV 31 and 18 E6 and E7, and host oncoproteins AKT, IQGAP1 and MMP16. Results: The model showed good performance, with an MCC score of 0.7937 and all other metrics above 88%. The model predicted an interaction between E6 and E7 of both HPV types with AKT, while only HPV31 E7 was shown to interact with IQGAP1 and MMP16 with confidence scores of 0.9638 and 0.5793, respectively. Conclusion: The current model strongly predicted HPVs E6 and E7 interactions with PI3K pathway, and the viral proteins may be involved in AKT activation, driving HPV-associated cancers. This model supports the robust prediction of interactomes for experimental validation. [ABSTRACT FROM AUTHOR]

The Epstein–Barr virus (EBV) is usually acquired during infancy as an asymptomatic infection and persists throughout life in a latent state under the control of the host immune system. However, EBV is associated with various malignant diseases that preferentially develop in immunodeficient individuals. Accumulating evidence suggests an important role for NK cells, though the mechanisms by which EBV evades or triggers NK cell responses are poorly understood. Here, we generated EBV-immortalized lymphoblastoid cell lines stably expressing an inducible form of the BZLF1 early lytic viral protein (LCL-Z) to challenge primary NK cells with EBV+ targets in either the latent or lytic phase of infection. We show that entry into the lytic phase results in drastic downregulation of HLA-E but not HLA-A, -B, and -C molecules and in increased expression of ligands for the activating NKG2D receptor, with MICB being upregulated at the cell membrane and released in a soluble form while ULBP2 and ULBP4 accumulate intracellularly. Furthermore, LCL-Z cells are killed by NK cells in an NKG2D-dependent manner and to a much higher extent during the lytic phase, but HLA-class I molecules constrain killing throughout the viral life cycle; unexpectedly, the antibody-mediated block of the inhibitory NKG2A receptor results in reduced lysis of lytic LCL-Z cells that are nearly devoid of the cognate HLA-E ligand. Accordingly, we show that NKG2A+ NK cell subsets, specifically CD56bright and NKG2A+KIR+CD56dim cells, are those that preferentially respond against cells with lytic EBV replication. Overall, these results shed light on NK/EBV+ cell interactions providing new information for improving NK cell-based immunotherapies to treat EBV-induced diseases. [ABSTRACT FROM AUTHOR]

Hantaviridae are a family of segmented negative-sense RNA viruses that contains important human and animal pathogens. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome. Here we establish the expression and purification polymerase from the Old World Hantaan virus and characterise the structure using Cryo-EM. We determine a series of structures at resolutions between 2.7 and 3.3 Å of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerases. We further describe structures with 5′ vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including β-sheet, two helical arrangements, and nucleotide primed arrangement. The insights gained here guide future mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets. Author summary: Hantaviruses are widely distributed viruses that can undergo zoonosis where they cause severe disease in humans. Old World hantaviruses, found predominantly in Asia and Europe, can cause a haemorrhagic fever with renal syndrome. Presently there are no approved specific treatments for infection and current vaccines are of limited efficacy. One of the viral encoded proteins called the polymerase has been identified as a good candidate for therapeutic development owing to its conservation, multiple enzymatic activities, and core function in the replication and transcription of the viral genome. Here we developed protocols to express highly pure polymerase and subsequently determined its three-dimensional structure in RNA-free, RNA-bound, and RNA/NTP bound states. We observe a full-length polymerase structure with the mobile domains arranged in a location not previously observed in either hantavirus polymerases or in polymerases from the wider bunyavirus family. Comparison of the polymerase core showed extensive rearrangement of a region called motif E and we observe the region in helical, sheet, and loop conformations. Using these models we are able to propose a scheme for the replication initiation. These findings are important in developing potential therapeutic inhibitors specific for hantaviruses. [ABSTRACT FROM AUTHOR]