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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.

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.

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.

Yaba monkey tumor virus (YMTV) and Tanapox virus (TPV) are members of the Yatapoxvirus genus and can infect humans and other primates. Despite the threat posed by yatapoxviruses, the factors determining their host range are poorly understood. In this study, we analyzed the ability of YMTV and TPV orthologs of vaccinia virus K3 (called 012 in YMTV and TPV), which share 75% amino acid identity with one another, to inhibit PKR from 15 different primate species. We first used a luciferase-based reporter, and found that YMTV and TPV K3 orthologs inhibited PKR in a species-specific manner and showed distinct PKR inhibition profiles. TPV 012 inhibited PKR from 11 primates, including humans, substantially better than YMTV 012. In contrast, both K3 orthologs inhibited the other four primate PKRs comparably well. Using YMTV 012 and TPV 012 hybrids, we mapped the region responsible for the differential PKR inhibition to the C- terminus of the K3 orthologs. Next, we generated chimeric vaccinia virus strains to investigate whether TPV K3 and YMTV K3 orthologs could rescue the replication of a vaccinia virus strain that lacks PKR inhibitors K3L and E3L. Virus replication in primate-derived cells generally correlated with the patterns observed in the luciferase-based assay. Together, these observations demonstrate that yatapoxvirus K3 orthologs have distinct PKR inhibition profiles and inhibit PKR in a species-specific manner, which may contribute to the differential susceptibility of primate species to yatapoxvirus infections.

The dengue virus (DENV) is a mosquito-borne flavivirus transmitted through the infected mosquito, primarily of the Aedes genus. Over recent decades, the significant emergence of DENV infections has posed growing challenges for control efforts. Extensive research indicates that the dengue NS2B–NS3 protease, a non-structural viral protein, is the most promising target in dengue drug research. Therefore, our study synthesized two series of novel allosteric inhibitors of the NS2B–NS3 protease to explore previous allosteric inhibitors of the protease. Meanwhile, enzyme inhibitory activities of all compounds are measured and IC50 values of the optimized compounds are obtained by utilizing a fluorescence-based assay. Notably, compound A24 demonstrates the highest enzyme inhibitory activity with an IC50 value of 16 μM. Further enzyme kinetic experiments confirm that the optimized compound A24 functions as a non-competitive inhibitor of the NS2B–NS3 protease. Through molecular docking studies, compound A24 showed a binding energy of −5.6 kcal mol−1, comparable to the lead compounds, which preliminarily explains its similar inhibitory activity. In addition, compound A24 forms a stable complex with the protein primarily through hydrogen bonds and π–π interaction, with subsequent dynamics simulations confirming the stable binding mode. In conclusion, we designed and synthesized 59 novel scaffold compounds and identified compound A24 with the highest activity through pharmacological experiments and computer-aided drug design, which targets the allosteric sites and contributes significantly to the prevention of the dengue virus. [ABSTRACT FROM AUTHOR]

Arboviruses, transmitted by medical arthropods, pose a serious health threat worldwide. During viral infection, Post Translational Modifications (PTMs) are present on both host and viral proteins, regulating multiple processes of the viral lifecycle. In this study, a mammalian E3 ubiquitin ligase WWP2 (WW domain containing E3 ubiquitin ligase 2) is identified, which interacts with the NS1 protein of Zika virus (ZIKV) and mediates K63 and K48 ubiquitination of Lys 265 and Lys 284, respectively. WWP2‐mediated NS1 ubiquitination leads to NS1 degradation via the ubiquitin‐proteasome pathway, thereby inhibiting ZIKV infection in mammalian hosts. Simultaneously, it is found Su(dx), a protein highly homologous to host WWP2 in mosquitoes, is capable of ubiquitinating NS1 in mosquito cells. Unexpectedly, ubiquitination of NS1 in mosquitoes does not lead to NS1 degradation; instead, it promotes viral infection in mosquitoes. Correspondingly, the NS1 K265R mutant virus is less infectious to mosquitoes than the wild‐type (WT) virus. The above results suggest that the ubiquitination of the NS1 protein confers different adaptations of ZIKV to hosts and vectors, and more importantly, this explains why NS1 K265‐type strains have become predominantly endemic in nature. This study highlights the potential application in antiviral drug and vaccine development by targeting viral proteins' PTMs. [ABSTRACT FROM AUTHOR]

Spike (S) glycoprotein is the largest structural protein of SARS‐CoV‐2 virus and the main one involved in anchoring of the host receptor ACE2 through the receptor binding domain (RBD). S protein secondary structure is of great interest for shedding light on various aspects, from functionality to pathogenesis, finally to spectral fingerprint for the design of optical biosensors. In this paper, the secondary structure of SARS‐CoV‐2 S protein and its constituting components, namely RBD, S1 and S2 regions, are investigated at serological pH by measuring their amide I infrared absorption bands through Attenuated Total Reflection Infrared (ATR‐IR) spectroscopy. Experimental data in combination with MultiFOLD predictions, Define Secondary Structure of Proteins (DSSP) web server and Gravy value calculations, provide a comprehensive understanding of RBD, S1, S2, and S proteins in terms of their secondary structure content, conformational order, and interaction with the solvent. [ABSTRACT FROM AUTHOR]

Introduction: The development of a potent immune response and antibodies against SARS-CoV-2 is important for herd immunity. The serological response may be due to a previous infection or vaccination. Healthy blood donors could represent and provide information on the immune status of the general population. Therefore, we estimated the global and regional prevalence of SARS-CoV-2 antibodies among healthy blood donors. Methods: We conducted a systematic search of PubMed, Scopus, and ProQuest from December 2019 to January 2023. After critical appraisal and quality assessment, a qualitative synthesis of the identified relevant articles was performed. The random-effects model was used to estimate the pooled prevalence of SARS-CoV-2 antibodies. Funnel plots and Egger's test were used to assess publication bias. Sensitivity analysis was performed, and heterogeneity was quantified using I2 statistics. Results: A total of 70 peer-reviewed articles were selected that together included 2,454,192 blood donors. The global estimated pooled prevalence of SARS-CoV-2 antibodies among healthy blood donors was 10.3% (95% CI: 4.6 – 18.0%, n = 70). The highest seroprevalence was observed in Asia (17.7%), followed by Africa (16.1%). The seroprevalence in studies conducted before the introduction of the vaccine was 6.1%, whereas those of studies conducted after vaccines were available was 27.6%. High seroprevalence was observed in studies that measured antibodies against the S protein of the virus (15.2%), while lower (12.5%) in those that measured antibodies against the N protein. A high seroprevalence was observed in studies that only measured IgG antibodies (16.3%), and it was 5.9% in studies that measured total antibodies. Conclusion: The prevalence of SARS-CoV-2 antibodies among healthy blood donors varies, potentially indicating geographical disparities in transmission and vaccination rates. To enhance community resilience, addressing these differences through inclusive health policies and adaptable public health measures is crucial. [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]

As a powerful eukaryotic expression vector, the baculovirus expression vector system (BEVS) is widely applied to the production of heterogeneous proteins for research and pharmaceutical purposes, while optimization of BEVS remains a work in progress for membrane or secreted protein expression. In this study, the impact of the signal peptide (SP) derived from Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 protein on protein expression, secretion, and the endoplasmic reticulum-associated degradation (ERAD) pathway were investigated in BmN cells and BEVS. Transient expression studies in BmN cells revealed that SP alters the localization and expression levels of recombinant proteins, reducing intracellular accumulation while enhancing secretion efficiency. Quantitative analysis demonstrated that SP-mediated secretion was markedly higher compared to controls, albeit with lower total expression levels. Further exploration into SP-mediated ERAD pathway activation showed increased expression of BiP and other ERAD-associated genes (PDI, UFD1, S1P, and ASK1), correlating with higher SP-driven protein expression levels. RNA interference (RNAi) experiments elucidated that knockdown of ERAD-associated genes enhances both the secretion efficiency of SP-guided proteins and the infectivity of BmNPV. Particularly, interference with BiP demonstrated the most pronounced effect on protein secretion enhancement. Viral infection experiments further supported these findings, showing upregulated ERAD-associated genes during BmNPV infection, indicating their role in viral protein processing and infectivity. In conclusion, this study elucidates the complex interplay between SP-mediated protein secretion, ERAD pathway activation, and viral infectivity in BmNPV-infected cells. These insights suggest strategies for optimizing recombinant protein production and viral protein processing in baculovirus expression systems, with potential implications for biotechnological and biomedical applications. Further research could refine our understanding and manipulation of protein secretion pathways in insect cell-based expression systems. [ABSTRACT FROM AUTHOR]

ELISA kits that detect antibodies to the non-structural protein (NSP) of the foot-and-mouth disease virus (FMDV), commonly referred to as NSP-ELISA, can distinguish between vaccinated and naturally infected animals. They can play an essential role in demonstrating 'proof-of-freedom' during the control of FMD. Although various NSP-ELISA kits are available in Thailand, information regarding their performance is lacking. To select the most appropriate NSP-ELISA kit for our specific purpose, we must compare their performance using carefully characterized sera. This will ensure that we maximize the benefits of our testing. In this study, six NSP-ELISA kits sold in Thailand―Biovet, ID Screen, VDPro, IDEXX, PrioCHECK, and KUcheck-F―were evaluated and compared. A total of 800 serum samples were examined, including samples from 357 cattle and 29 buffaloes in outbreak areas, as well as 14 swine serum samples from the Vaccine Quality Control Unit of the Bureau of Veterinary Biologics, Ministry of Agriculture and Cooperation, Thailand. Four hundred samples were confirmed to originate from animals infected with FMDV through ELISA typing (n = 11, tested as representative samples in each farm) and/or RT-PCR (n = 400, all samples), serving as positive control sera. Additionally, 400 negative control sera were obtained from Japan (97 cattle and 300 pigs) and Australia (3 goats), certified by the World Organisation for Animal Health as 'free of FMD'. The sensitivity and specificity of the six tests were determined based on the results obtained from two-by-two tables. Cohen's kappa statistics were calculated for the six tests to assess their concordance, and the diagnostic accuracy of the assays was also determined. For all six NSP-ELISA kits, the sensitivity ranged from 97.75 to 99.50%, and the specificity ranged from 97.25 to 100%. Cohen's kappa statistics ranged from 0.96 to 1.00, and diagnostic accuracy ranged from 98.13 to 99.75%. The study results indicated that the test kits have statistically similar sensitivity, specificity, concordance, and diagnostic accuracy, suggesting they can be used interchangeably. However, ID Screen demonstrated the highest sensitivity and specificity among all kits tested. Therefore, if a single kit were to be selected from the six evaluated, ID Screen would be the most appropriate choice. These findings can aid in selecting the most suitable test kit. Therefore, it is recommended to consider purchasing a diverse range of effective test kits. Furthermore, these findings can provide guidance for expanding the use of test kits, particularly with the growing availability of NSP-ELISA kits in the market. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) infection, one of the most common forms of hepatitis worldwide, is often associated with extrahepatic, particularly renal, manifestations. However, the underlying mechanisms are incompletely understood. Here, we report the development of a de novo immune complex-mediated glomerulonephritis (GN) in a kidney transplant recipient with chronic hepatitis E. Applying immunostaining, electron microscopy, and mass spectrometry after laser-capture microdissection, we show that GN develops in parallel with increasing glomerular deposition of a non-infectious, genome-free and non-glycosylated HEV open reading frame 2 (ORF2) capsid protein. No productive HEV infection of kidney cells is detected. Patients with acute hepatitis E display similar but less pronounced deposits. Our results establish a link between the production of HEV ORF2 protein and the development of hepatitis E-associated GN in the immunocompromised state. The formation of glomerular IgG-HEV ORF2 immune complexes discovered here provides a potential mechanistic explanation of how the hepatotropic HEV can cause variable renal manifestations. These findings directly provide a tool for etiology-based diagnosis of hepatitis E-associated GN as a distinct entity and suggest therapeutic implications. It's poorly understood how renal manifestations develop in HEV infection in patients. Here, the authors observe glomerular accumulation of the viral protein ORF2 in complex with host IgG in immunocompromised patients with chronic or acute HEV infection who developed glomerulonephritis. [ABSTRACT FROM AUTHOR]

In the past 25 years, the world has witnessed outbreaks of illnesses in humans from three different coronaviruses. Both the SARS-CoV outbreak of 2003 and the MERS-CoV outbreak of 2013 resulted in overall low fatalities in part due to inefficient human-to-human spread of each virus. In contrast, SARS-CoV-2, which emerged in 2019, was highly efficient at human-to-human spread and caused a global pandemic resulting in millions of casualties. Zoonotic transmission of viruses, including the three coronaviruses, poses an ongoing threat that cannot be ignored. In this review, we have focused on the diagnostics and therapeutics fronts using SARS-CoV-2 as a model. Specifically, we have selected proteins associated with the virus particles as targets and discussed various platform technologies. These insights hold the potential to inform the development of more effective therapeutics and vaccines not only for SARSCoV-2 but also for future viral pandemics, thus contributing to global health on a broader scale. [ABSTRACT FROM AUTHOR]

Non-POU domain-containing octamer-binding protein (NONO) is a multi-functional nuclear protein which belongs to the Drosophila behavior/human splicing (DBHS) protein family. NONO is known to regulate multiple important biological processes including host antiviral immune response. However, whether NONO can inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication is less well understood. In this study, we demonstrated that swine NONO (sNONO) inhibited PRRSV replication, via increasing expression of IFN-β, whereas NONO knockdown or knockout in PAM-KNU cells was more susceptible to PRRSV infection. As an IRF3 positive regulation factor, NONO promoted IFN-β expression by enhancing activation of IRF3. During PRRSV infection, NONO further up-regulated IRF3-mediated IFN-β expression by interacting with PRRSV N protein. Mechanistically, NONO functioned as a scaffold protein to detect PRRSV N protein and formed N-NONO-IRF3 complex in the nucleus. Interestingly, it was found that the NONO protein reversed the inhibitory effect of PRRSV N protein on type I IFN signaling pathway. Taken together, our study provides a novel mechanism for NONO to increase the IRF3-mediated IFN-β activation by interacting with the viral N protein to inhibit PRRSV infection. Author summary: Porcine reproductive and respiratory syndrome (PRRS), which is caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a highly infectious disease and leads to huge economic losses to the global swine industry. Here, we reported that Non-POU domain-containing octamer-binding protein (NONO) significantly inhibited PRRSV replication and increased the expression of IFN-β. We found that NONO positively regulated IFN-β signaling pathway by targeting IRF3 and interacted with PRRSV N protein to promote activation of IFN-β, which reversed the inhibitory effect of PRRSV N protein on type I IFN signaling. We also found that NONO acted as a bridge to recruit PRRSV N and IRF3 to form N-NONO-IRF3 complex and enhanced activation of IFN-β signaling. Our study uncovers a novel mechanism for NONO to exert antiviral response through interacting with PRRSV N protein and IRF3 to inhibit PRRSV replication. [ABSTRACT FROM AUTHOR]

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production. Author summary: Chikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by mosquitoes. Its replication relies on a polymerase that incorporates a significant number of errors in the new genomes, making it a good candidate to develop vaccines or antiviral strategies. However, little is known on alphavirus polymerase function in alternate hosts. To begin to understand how the CHIKV polymerase nsP4 functions, we designed a panel of nsP4 variants taking advantage of the conservation of polymerase structure across positive-strand RNA viruses. We discovered that the palm domain and ring finger subdomain of the polymerase were involved in host-specific RNA transcription and virion production. Taken together, these findings add further evidence to the crucial impact of the core palm domain of CHIKV polymerase not only on the transcription of the RNA itself, but also on the complete viral life cycle. [ABSTRACT FROM AUTHOR]

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host's antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host's innate antiviral immunity and thus provides insights into viral pathogenesis. [ABSTRACT FROM AUTHOR]

The proteasome plays a crucial role in cellular homeostasis by degrading misfolded, damaged, or unnecessary proteins. Understanding the regulatory mechanisms of proteasome activity is vital, particularly the interaction with activators containing the hydrophobic-tyrosine-any amino acid (HbYX) motif. Here, we present ProEnd, a comprehensive database designed to identify and catalog HbYX motif-containing proteins across the tree of life. Using a simple bioinformatics pipeline, we analyzed approximately 73 million proteins from 22,000 reference proteomes in the UniProt/SwissProt database. Our findings reveal the widespread presence of HbYX motifs in diverse organisms, highlighting their evolutionary conservation and functional significance. Notably, we observed an interesting prevalence of these motifs in viral proteomes, suggesting strategic interactions with the host proteasome. As validation two novel HbYX proteins found in this database were experimentally tested by pulldowns, confirming that they directly interact with the proteasome, with one of them directly activating it. ProEnd's extensive dataset and user-friendly interface enable researchers to explore the potential proteasomal regulator landscape, generating new hypotheses to advance proteasome biology. This resource is set to facilitate the discovery of novel therapeutic targets, enhancing our approach to treating diseases such as neurodegenerative disorders and cancer. [ABSTRACT FROM AUTHOR]

ABSTRACT The human papillomavirus (HPV) 33 is a high‐risk strain that causes lesions with potential cancerous outcomes. Its E2 protein regulates the viral protein transcription and life cycle maintenance. The DNA binding domain (DBD) of the E2 protein plays a crucial role in the viral life cycle. The DBD region of the E2 protein is particularly interesting for targeting and finding potential inhibitors to inhibit its function or dimerization. Given the limited research on HPV 33 and its proteins, the present work delved into the interaction of two natural polyphenolic compounds, resveratrol, and baicalein, with the E2 DBD of HPV 33 using biophysical and in silico studies. Fluorescence studies of the E2 DBD‐polyphenol complexes showed fluorescence quenching with a binding constant of the order of 106 M−1. Circular dichroism data reveal conformational changes upon binding with the polyphenols, possibly due to distinct binding sites of the E2 DBD. Differential scanning calorimetry exhibited higher melting temperatures for the two complexes than alone DBD, suggesting the complexes' stability. ITC experiment suggested favorable binding reactions with kd values in the micromolar range. Molecular docking and dynamic simulation studies revealed that the resveratrol binds to the helical region and baicalein near the central dimeric interface of E2 DBD with a good binding affinity, forming a stable protein‐ligand complex during the run of 100 ns simulation. Therefore, the current study identifies both polyphenolic compounds as promising candidates for potential antiviral drug development. [ABSTRACT FROM AUTHOR]

Introduction: The frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear. Methods: We designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptidekeyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund's adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay. Results: Seroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomainbased vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro. Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30. Discussion: The findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections. [ABSTRACT FROM AUTHOR]

ASFV C315R is homologous to the transcription factor TFIIB of large unclassified DNA viruses, and H359L is identical to the subunit 3 (RPB3) of eukaryotic RNA polymerase II. The C315R and H359L may play an important role in ASFV replication and transcription. Here, we evaluated the biological function of the C315R and H359L genes during virus replication in vitro and during infection in pigs. Results showed that C315R and H359L are highly conserved among ASFV genotype II strains; quantitative PCR (qPCR) and western blotting analyses revealed that C315R and H359L are early transcribed genes prior to viral DNA replication, but their protein expression is delayed. The immunofluorescence and western blotting analysis revealed that both proteins localized in the cell cytoplasm and nucleus at 24 h post infection, however, pH359L was mainly detected in the cell cytoplasm. Furthermore, overexpression of pH359L in MA104 cells significantly increased viral titer, RNA transcription levels, and viral protein expression levels, while overexpression of pC315R slightly enhanced ASFV replication. In contrast, siRNA targeting ASFV-H359L or C315R reduced replication efficiency in porcine macrophage culture compared to the parent ASFV-CN/SC/2019, demonstrating that C315R and H359L genes are necessary for ASFV replication. Finally, the functional role of C315R or H359L on PKR and eIF2a phosphorylation status and SG formation, as well as cytokine production were evaluated. These studies demonstrated that C315R and H359L are involved in virus replication processes in swine and play important roles in ASFV replication. [ABSTRACT FROM AUTHOR]

Enterovirus (EV) infections occur frequently in humans. In some geographical areas they are more common. These viruses cause diseases with varying degrees of severity, from a simple respiratory tract infection to severe diseases. Since EVs include more than 70 serotypes currently circulating in the population, a methodology that detects most of them is needed. ELISA is a rapid, sensitive, and economical diagnostic method for the identification of EV serotypes and can also be used as a retrospective diagnostic tool or in the investigation of outbreaks of infection. Commercial EV-ELISAs often appear and gradually disappear from the market supply. We have used the KTL-510 peptide, a synthetic viral protein of poliovirus VP1, as an antigen in a peptide-based ELISA for the detection of a broader spectrum of anti-EV antibodies. We aimed to design, optimize, and standardize this in-house ELISA with the peptide, and implement the method for routine detection of anti-EV IgG in human sera. For determining the cut-off value, we used 100 patients' sera which were previously tested negative for IgG antibodies against EVs using a commercial ELISA kit available. We monitored patients' sera samples sent for serological testing of anti-coxsackievirus antibodies to the National Reference Center for the Identification of Enteric Viruses between 2018-2022. These serum samples were examined using a standard virus neutralization test as well as the newly developed ELISA method. [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]

Various viral proteins are post-translationally modified by SUMO-conjugation during the human adenovirus (HAdV) replication cycle. This modification leads to diverse consequences for target proteins as it influences their intracellular localization or cell transformation capabilities. SUMOylated HAdV proteins include the multifunctional oncoprotein E1B-55K. Our previous research, along with that of others, has demonstrated a substantial influence of yet another adenoviral oncoprotein, E4orf6, on E1B-55K SUMOylation levels. Protein SUMOylation can be reversed by cellular sentrin/SUMO-specific proteases (SENPs). In this study, we investigated the interaction of E1B-55K with cellular SENPs to understand deSUMOylation activities and their consequences for cell transformation mediated by this adenoviral oncoprotein. We show that E1B-55K interacts with and is deSUMOylated by SENP 1, independently of E4orf6. Consistent with these results, we found that SENP 1 prevents E1A/E1Bdependent focus formation in rodent cells. We anticipate these findings to be the groundwork for future studies on adenovirus-host interactions, the mechanisms that underlie E1B-55K SUMOylation, as well as the role of this major adenoviral oncoprotein in HAdV-mediated cell transformation. [ABSTRACT FROM AUTHOR]

Because host kinases are key regulators of multiple signaling pathways in response to viral infections, we previously screened a kinase inhibitor library using rhabdomyosarcoma cells and human intestinal organoids in parallel to identify potent inhibitors against EV-A71 infection. We found that Rho-associated coiled-coil-containing protein kinase (Rock) inhibitor efficiently suppressed the EV-A71 replication and further revealed Rock1 as a novel EV-A71 host factor. In this study, subsequent analysis found that a variety of vascular endothelial growth factor receptor (VEGFR) inhibitors also had potent antiviral effects. Among the hits, Pazopanib, with a selectivity index as high as 254, which was even higher than that of Pirodavir, a potent broad-spectrum picornavirus inhibitor targeting viral capsid protein VP1, was selected for further analysis. We demonstrated that Pazopanib not only efficiently suppressed the replication of EV-A71 in a dose-dependent manner, but also exhibited broad-spectrum anti-enterovirus activity. Mechanistically, Pazopanib probably induces alterations in host cells, thereby impeding viral genome replication and transcription. Notably, VEGFR2 knockdown and overexpression suppressed and facilitated EV-A71 replication, respectively, indicating that VEGFR2 is a novel host dependency factor for EV-A71 replication. Transcriptome analysis further proved that VEGFR2 potentially plays a crucial role in combating EV-A71 infection through the TSAd-Src-PI3K-Akt pathway. These findings expand the range of potential antiviral candidates of anti-enterovirus therapeutics and suggest that VEGFR2 may be a key host factor involved in EV-A71 replication, making it a potential target for the development of anti-enterovirus therapeutics. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) belongs to the family of Picornaviridae; it could cause a variety of illnesses and pose a great threat to public health worldwide. Currently, there is no specific drug treatment for this virus, and a better understanding of virushost interaction is crucial for novel antiviral development. Here, we find that the class III phosphatidylinositol 3-kinase, VPS34, is an essential host factor for EV71 infection. VPS34 inhibition with either shRNA or specific chemical inhibitor significantly reduces EV71 infection. Meanwhile, EV71 infection upregulates phosphatidylinositol 3-phosphate (PI3P) production in viral replication organelles (ROs), while the depletion of PI3P by phosphatase overexpression inhibits EV71 infection. In addition, the PI3P-binding protein, double FYVE-containing protein 1 (DFCP1), is also required for an efficient replication of EV71. DFCP1 could interact with viral 2C protein and facilitate viral association with lipid droplets (LDs), which are important lipid sources for viral RO biogenesis. Taken together, these results indicate that EV71 virus exploits the VPS34- PI3P-DFCP1-LDs pathway to promote viral RO formation and viral infection, and they also illuminate novel targets for antiviral development. [ABSTRACT FROM AUTHOR]

Transcriptional activity of RNA polymerase II (Pol II) is influenced by post-translational modifications of the C-terminal domain (CTD) of the largest Pol II subunit, RPB1. Herpes simplex virus type 1 (HSV-1) usurps the cellular transcriptional machinery during lytic infection to efficiently express viral mRNA and shut down host gene expression. The viral immediate-early protein ICP22 interferes with serine 2 phosphorylation (pS2) by targeting CDK9 and other CDKs, but the full functional implications of this are not well understood. Using Western blotting, we report that HSV-1 also induces a loss of serine 7 phosphorylation (pS7) of the CTD during lytic infection, requiring expression of the two immediate-early proteins ICP22 and ICP27. ICP27 has also been proposed to target RPB1 for degradation, but we show that pS2/S7 loss precedes the drop in total protein levels. Cells with the RPB1 polyubiquitination site mutation K1268R, preventing proteasomal degradation during transcription-coupled DNA repair, displayed loss of pS2/S7 but retained higher overall RPB1 protein levels later in infection, indicating this pathway is not involved in early CTD dysregulation but may mediate bulk protein loss later. Using α-amanitin-resistant CTD mutants, we observed differential requirements for Ser2 and Ser7 for the production of viral proteins, with Ser2 facilitating viral immediate-early genes and Ser7 appearing dispensable. Despite dysregulation of CTD phosphorylation and different requirements for Ser2/7, all CTD modifications tested could be visualized in viral replication compartments with immunofluorescence. These data expand the known means that HSV employs to create pro-viral transcriptional environments at the expense of host responses. [ABSTRACT FROM AUTHOR]

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles. [ABSTRACT FROM AUTHOR]

The respiratory syncytial virus (RSV) matrix (M) protein plays an important role in infection as it can interact with viral components as well as the host cell actin microfilaments. The M-actin interaction may play a role in facilitating the transportation of virion components to the apical surface, where RSV is released. We show that M protein's association with actin is facilitated by palladin, an actin-binding protein. Cells were infected with RSV or transfected to express full-length M as a green fluorescent protein (GFP)-tagged protein, followed by removal of nuclear and cytosolic proteins to enrich for cytoskeleton and its associated proteins. M protein was present in inclusion bodies tethered to microfilaments in infected cells. In transfected cells, GFP-M was presented close to microfilaments, without association, suggesting the possible involvement of an additional protein in this interaction. As palladin can bind to proteins that also bind actin, we investigated its interaction with M. Cells were co-transfected to express GFP-M and palladin as an mCherry fluorescenttagged protein, followed by cytoskeleton enrichment. M and palladin were observed to colocalize towards microfilaments, suggesting that palladin is involved in the M-actin interaction. In co-immunoprecipitation studies, M was found to associate with two isoforms of palladin, of 140 and 37 kDa. Interestingly, siRNA downregulation of palladin resulted in reduced titer of released RSV, while cell associated RSV titer increased, suggesting a role for palladin in virus release. Together, our data show that the M-actin interaction mediated by palladin is important for RSV budding and release. [ABSTRACT FROM AUTHOR]

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus that can cause several cancers, such as Kaposi sarcoma and primary effusion lymphoma (PEL). We and others have recently demonstrated that Forkhead box (FOX) transcription factors can be dysregulated by KSHV, and they can affect KSHV infection. Herein, we focus on dissecting the role of two FOXK subfamily members, FOXK1 and FOXK2, in the KSHV life cycle. FOXK proteins are key host regulators of cellular functions, yet their role in KSHV infection remains unknown. Here, we demonstrated that both FOXK proteins are essential for efficient KSHV lytic reactivation in PEL cells. FOXK1 and FOXK2 are unique as they are the only FOX proteins that contain a Forkhead-associated (FHA) domain. The FHA domain is a specialized protein binding domain that recognizes a short linear serine/threonine-rich (S/T) motif. Through an unbiased motif survey, we found that KSHV viral protein ORF45 and its gammaherpesvirus homologs contain a putative FHA-binding motif. ORF45 is an immediate early tegument protein, vital for lytic reactivation and virus production. We demonstrated that ORF45 uses its novel conserved motif to interact with the FHA domain containing FOXK factors in the nucleus of infected cells. A single-point mutation of the conserved threonine residue in the motif within ORF45 abolished the ORF45-FOXK1/2 interaction. Our data indicates that FOXK proteins interact with ORF45 homologs encoded by murine gammaherpesvirus 68 (MHV68) and Rhesus macaque rhadinovirus (RRV), and that the FHA domains of FOXK proteins are sufficient for their interactions, highlighting a conserved mechanism. [ABSTRACT FROM AUTHOR]

Selective autophagy is a protein clearance mechanism mediated by evolutionarily conserved selective autophagy receptors (SARs), which specifically degrades misfolded, misassembled, or metabolically regulated proteins. SARs help the host to suppress viral infections by degrading viral proteins. However, viruses have evolved sophisticated mechanisms to counteract, evade, or co-opt autophagic processes, thereby facilitating viral replication. Therefore, this review aims to summarize the complex mechanisms of SARs involved in viral infections, specifically focusing on how viruses exploit strategies to regulate selective autophagy. We present an updated understanding of the various critical roles of SARs in viral pathogenesis. Furthermore, newly discovered evasion strategies employed by viruses are discussed and the ubiquitination-autophagy-innate immune regulatory axis is proposed to be a crucial pathway to control viral infections. This review highlights the remarkable flexibility and plasticity of SARs in viral infections. [ABSTRACT FROM AUTHOR]

Negevirus is a recently proposed taxon of arthropod-infecting virus, which is associated with plant viruses of two families (Virgaviridae and Kitaviridae). Nevertheless, the evolutionary history of negevirus-host and its relationship with plant viruses remain poorly understood. Endogenous nege-like viral elements (ENVEs) are ancient nege-like viral sequences integrated into the arthropod genomes, which can serve as the molecular fossil records of previous viral infection. In this study, 292 ENVEs were identified in 150 published arthropod genomes, revealing the evolutionary history of nege-like viruses and two related plant virus families. We discovered three novel and eight strains of nege-like viruses in 11 aphid species. Further analysis indicated that 10 ENVEs were detected in six aphid genomes, and they were divided into four types (ENVE1-ENVE4). Orthologous integration and phylogenetic analyses revealed that nege-like viruses had a history of infection of over 60 My and coexisted with aphid ancestors throughout the Cenozoic Era. Moreover, two nege-like viral proteins (CP and SP24) were highly homologous to those of plant viruses in the families Virgaviridae and Kitaviridae. CP- and SP24-derived ENVEs were widely integrated into numerous arthropod genomes. These results demonstrate that nege-like viruses have a long-term coexistence with arthropod hosts and plant viruses of the two families, Virgaviridae and Kitaviridae, which may have evolved from the nege-like virus ancestor through horizontal virus transfer events. These findings broaden our perspective on the history of viral infection in arthropods and the origins of plant viruses. [ABSTRACT FROM AUTHOR]

Ubiquitin modification of viral proteins to degrade or regulate their function is one of the strategies of the host to resist viral infection. Here, we report that ubiquitin protein ligase E3C (UBE3C), an E3 ubiquitin ligase, displayed inhibitory effects on EV-A71 replication. UBE3C knockdown resulted in increased viral protein levels and virus titers, whereas overexpression of UBE3C reduced EV-A71 replication. To explore the mechanism by which UBE3C affected EV-A71 infection, we found that the C-terminal of UBE3C bound to 2C protein and facilitated K33/K48-linked ubiquitination degradation of 2C K268. Moreover, UBE3C lost its ability to degrade 2C K268R and had a diminished inhibitory impact against the replication of recombinant EV-A71-FY-2C K268R. In addition, UBE3C also promoted ubiquitination degradation of the 2C protein of CVB3 and CVA16 and inhibited viral replication. Thus, our findings reveal a novel mechanism that UBE3C acts as an enterovirus host restriction factor, including EV-A71, by targeting the 2C protein. [ABSTRACT FROM AUTHOR]

Cyclosporin A (CsA), an immunosuppressive drug used in transplant recipients, inhibits graft rejection by binding to cyclophilins and competitively inhibiting calcineurin. While concerns about respiratory infections in immunosuppressed patients exist, contradictory data emerged during the COVID‐19 pandemic, prompting investigations into CsA's impact on viral infections. This study explores CsA's antiviral effects on SARS‐CoV‐2 Omicron BA.1, Delta variants, and human parainfluenza virus 3 (HPIV3) using an ex vivo model of human airway epithelium (HAE). CsA exhibited a dose‐dependent antiviral effect against the SARS‐CoV‐2 Delta variant, reducing viral load over 10 days. However, no significant impact was observed against SARS‐CoV‐2 Omicron or HPIV3, indicating a virus‐specific effect. At high concentrations, CsA was associated with an increase of IL‐8 and a decrease of IFNλ expression in infected and noninfected HAE. This study highlights the complexity of CsA's antiviral mechanisms, more likely involving intricate inflammatory pathways and interactions with specific viral proteins. The research provides novel insights into CsA's effects on respiratory viruses, emphasizing the need for understanding drug–virus interactions in optimizing therapeutic approaches for transplant recipients and advancing knowledge on immunosuppressive treatments' implications on respiratory viral infections. Limitations include the model's inability to assess T lymphocyte activation, suggesting the necessity for further comprehensive studies to decipher the intricate dynamics of immunosuppressive treatments on respiratory viral infections. [ABSTRACT FROM AUTHOR]

Eight porcine parvovirus (PPV) species, designated as PPV1 through PPV8, have been identified in swine. Despite their similarities, knowledge about their distribution and genetic differences remains limited, resulting in a gap in the genetic classification of these viruses. In this study, we conducted a comprehensive analysis using PPV1 to PPV7 genome sequences from Colombia and others available in the GenBank database to propose a classification scheme for all PPVs. Sera from 234 gilts aged 180 to 200 days were collected from 40 herds in Colombia. Individual detection of each PPV (PPV1 through PPV7) was performed using end-point PCR. Complete nucleotide (nt) sequencing was performed on the PPV1 viral protein (VP), and near-complete genome (NCG) sequencing was carried out for novel porcine parvoviruses (nPPVs) (PPV2 through PPV7). Phylogenetic analyses were conducted by comparing PPV1-VP sequences to 94 available sequences and nPPVs with 565 NCG, 846 nPPV-VP, and 667 nPPV–nonstructural protein (NS) sequences. Bayesian phylogenetic analysis was used to estimate substitution rates and the time to the most recent common ancestor for each PPV. The highest prevalence was detected for PPV3 (40.1%), followed by PPV5 (20.5%), PPV6 (17%), PPV1 (14.5%), PPV2 (9.8%), PPV4 (4.2%), and PPV7 (1.3%). Notably, all tested sera were negative for PPV8 genomes. An analysis of the PPV1-VP sequences revealed two main clades (PPV1-I and PPV1-II), with the sequences recovered in this study grouped in the PPV1-II clade. Comparative analysis showed significant genetic distances for PPV2 to PPV7 at the NCG (>6.5%), NS (>6.3%), and VP (>7.5%) regions, particularly when compared to equivalent regions of PPV genomes recovered worldwide. This study highlights the endemic circulation of nPPVs in Colombian pig herds, specifically among gilts. Additionally, it contributes to the phylogenetic classification and evolutionary studies of these viruses. The proposed method aims to categorize and divide subtypes based on current knowledge and the genomes available in databanks. [ABSTRACT FROM AUTHOR]

The severity of respiratory syncytial virus (RSV) may be linked to host genetic susceptibility. Surfactant protein (SP) genetic variants have been associated with RSV severity, but the impact of single-nucleotide polymorphism (SNP)-SNP interactions remains unexplored. Therefore, we used a novel statistical model to investigate the association of SNP-SNP interactions of SFTP genes with RSV severity in two- and three-interaction models. We analyzed available genotype and clinical data from prospectively enrolled 405 children diagnosed with RSV, categorizing them into moderate or severe RSV groups. Using Wang's statistical model, we studied significant associations of SNP-SNP interactions with RSV severity in a case-control design. We observed, first, association of three interactions with increased risk of severe RSV in a two-SNP model. One intragenic interaction was between SNPs of SFTPA2, and the other two were intergenic, involving SNPs of hydrophilic and hydrophobic SPs alone. We also observed, second, association of 22 interactions with RSV severity in a three-SNP model. Among these, 20 were unique, with 12 and 10 interactions associated with increased or decreased risk of RSV severity, respectively, and included at least one SNP of either SFTPA1 or SFTPA2. All interactions were intergenic except one, among SNPs of SFTPA1. The remaining interactions were either among SNPs of hydrophilic SPs alone (n = 8) or among SNPs of both hydrophilic or hydrophobic SPs (n = 11). Our findings indicate that SNPs of all SFTPs may contribute to genetic susceptibility to RSV severity. However, the predominant involvement of SFTPA1 and/or SFTPA2 SNPs in these interactions underscores their significance in RSV severity. NEW & NOTEWORTHY: Although surfactant protein (SP) genetic variants are associated with respiratory syncytial virus (RSV) severity, the impact of single-nucleotide polymorphism (SNP)-SNP interactions of SP genes remained unexplored. Using advanced statistical models, we uncovered 22 SNP-SNP interactions associated with RSV severity, with notable involvement of SFTPA1 and SFTPA2 SNPs. This highlights the comprehensive role of all SPs in genetic susceptibility to RSV severity, shedding light on potential avenues for targeted interventions. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has prompted the medical systems of many countries to develop effective treatments to combat the high rate of infection and death caused by the disease. Within the array of proteins found in SARS-CoV-2, the 3 chymotrypsin-like protease (3CLpro) holds significance as it plays a crucial role in cleaving polyprotein peptides into distinct functional nonstructural proteins. Meanwhile, RNA-dependent RNA polymerase (RdRp) takes center stage as the key enzyme tasked with replicating the viral genomic RNA within host cells. These proteins, 3CLpro and RdRp, are deemed optimal subjects for QSAR modeling due to their pivotal functions in the viral lifecycle. In this study, SMILES-based QSAR classification models were developed for a dataset of 2377 compounds that were defined as either active or inactive against 3CLpro and RdRp. Pharmacophore (PH4) and QSAR modeling were used for the virtual screening on 60.2 million compounds including ZINC, ChEMBL, Molport, and MCULE databases to identify new potent inhibitors against 3CLpro and RdRp. Then, a filter was established based on typical molecular characteristics to identify drug-like molecules. The molecular docking was also performed to evaluate the binding affinity of 156 AND 51 potential inhibitors to 3CLpro and RdRp, respectively. Among the 15 hits identified based on molecular docking scores, M3, N2, and N4 were identified as promising inhibitors due to their good synthetic accessibility scores (3.07, 3.11, and 3.29 out of 10 for M3, N2, and N4 respectively). These compounds contain amine functional groups, which are known for their crucial role in the binding interactions between drugs and their targets. Consequently, these hits have been chosen for further biological assay studies to validate their activity. They may represent novel 3CLpro and RdRp inhibitors possessing drug-like properties suitable for COVID-19 therapy. [ABSTRACT FROM AUTHOR]

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca2+) binding in the low-affinity Ca2+-binding pocket of NA, enhancing enzymatic activity and thermostability under Ca2+-depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca2+ binding in the low-affinity calcium-binding pocket. Modulation of calcium binding in the neuraminidase low-affinity calcium-binding pocket suggests a novel role of calcium during cross-species transmission of Influenza A viruses. [ABSTRACT FROM AUTHOR]

Carbohydrate binding modules (CBMs) are protein domains that typically reside near catalytic domains, increasing substrate-protein proximity by constraining the conformational space of carbohydrates. Due to the flexibility and variability of glycans, the molecular details of how these protein regions recognize their target molecules are not always fully understood. Computational methods, including molecular docking and molecular dynamics simulations, have been employed to investigate lectin-carbohydrate interactions. In this study, we introduce a novel approach that integrates multiple computational techniques to identify the critical amino acids involved in the interaction between a CBM located at the tip of bacteriophage J-1's tail and its carbohydrate counterparts. Our results highlight three amino acids that play a significant role in binding, a finding we confirmed through in vitro experiments. By presenting this approach, we offer an intriguing alternative for pinpointing amino acids that contribute to protein-sugar interactions, leading to a more thorough comprehension of the molecular determinants of protein-carbohydrate interactions. [ABSTRACT FROM AUTHOR]

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome–lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS. [Display omitted] • MBP interacts with SARS-CoV-2 proteins N, E, nsp9 and nsp16 in mammalian cells. • MBP interacts and colocalizes with SARS-CoV-2 protein ORF3a in mammalian cells. • MBP counteracts ORF3a-mediated failure of the autophagy machinery. • Basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition. [ABSTRACT FROM AUTHOR]

This work implements a method for specific in vivo biotinylation of recombinant proteins M1 and B7 of the variola virus during biosynthesis in CHO-K1 cells. To do this, co-expression of the biotin ligase BirA and target genes encoding the ectodomains of the M1 and B7 proteins with a C-terminal avi-tag was carried out in CHO-K1 cells in the presence of biotin in the culture medium. The optimal biotin concentration for the expression of the M1 and B7 proteins was 125 μM. The production of biotinylated recombinant proteins has been complicated by low yields. To increase the production of target proteins, low molecular weight enhancers were added to the culture medium: lithium acetate, sodium valproate, and caffeine. The enhancers increased the yield of the target protein by 1.3–4.9 times and did not affect the efficiency of biotinylation. The highest yield of biotinylated protein was achieved with the simultaneous addition of a concentration of 10 mM lithium acetate and 2.5 mM sodium valproate. [ABSTRACT FROM AUTHOR]

Dengue virus, an arbovirus of genus flavivirus, is one of the most prevalent infectious disease causing organisms in the tropical environment leading to numerous deaths every year. The envelope protein plays a crucial role in the viral genome entry into the host cell. This is achieved through structure and oligomeric status change in the dengue envelope protein. The pre-fusion and post-fusion states' structures of the dengue envelope protein are known although the pathway and intermediate structures involved in this process have remained in the dark till date. In this study, we have used targeted molecular dynamics (MD) simulation followed by conventional MD simulation and the Markov state model analysis to elucidate this pathway of change in conformation from the pre-fusion state to the post-fusion state. Intermediate conformations which may in the future serve as therapeutic targets have also been deduced from the Markov state model. This pathway determination may pave the way for the design of transition state analog inhibitors which prevent the dengue virus envelope protein from changing its conformation and thus establishing infection. The results obtained could lead to development of novel drugs to combat the viral infection. [ABSTRACT FROM AUTHOR]

Nuclear factor κB (NF‐κB) plays a crucial role in various cellular processes, including inflammatory and immune responses. Its activation is tightly regulated by the IKK (IκB kinase) complex. Upon severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, the virus is initially recognized by the innate immune system and typically activates the NF‐κB pathway, leading to a severe inflammatory response. However, the influence of viral proteins upon pro‐inflammatory pathway is complicated. Here, we demonstrated that the viral protein nsp3 of SARS‐CoV‐2 exhibits an unusual function, which attenuated the NF‐κB‐mediated inflammatory response against SARS‐CoV‐2 infection in a unique manner. nsp3 interacted with the essential NF‐κB modulator NEMO/IKKγ and promoted its polyubiquitylation via the E3 ubiquitin ligase CBL (Cbl Proto‐Oncogene). Consequently, polyubiquitylated NEMO undergoes proteasome‐dependent degradation, which disrupts NF‐κB activation. Moreover, we found that the SARS unique domain (SUD) in nsp3 of SARS‐CoV‐2 is essential for inducing NEMO degradation, whereas this function is absent in SUD of SARS‐CoV. The reduced activation of pro‐inflammatory response at an early stage could mask the host immune response and faciliate excessive viral replication. Conversely, this finding may partially explain why SARS‐CoV‐2 causes a less inflammatory reaction than SARS‐CoV, resulting in more mild or moderate COVID‐19 cases and greater transmissibility. Given that NEMO is important for NF‐κB activation, we propose that inhibiting polyubiquitylation and degradation of NEMO upon SARS‐CoV‐2 infection is a novel strategy to modulate the host inflammatory response. [ABSTRACT FROM AUTHOR]

BACKGROUND: Telosma mosaic virus (TelMV, Potyvirus, Potyviridae) is an emerging viral pathogen that threatens passion fruit plantations worldwide. However, an efficient strategy for controlling such a virus is not yet available. Cross protection is a phenomenon in which pre‐infection of a plant with one mild strain prevents or delays subsequent infection by the same or closely related virus. HC‐Pro is the potyviral encoded multifunctional protein involved in several steps of viral infection, including multiplication, movement, transmission and RNA silencing suppression. In this study, we tested whether it is possible to generate attenuated viral strains capable of conferring protection against severe TelMV infection by manipulating the HC‐Pro gene. RESULTS: By introducing point mutation into the conserved motif FRNK of HC‐Pro that is essential for RNA silencing suppression, we have successfully obtained three attenuated mutants of TelMV (R181K, R181D, and R181E, respectively). These attenuated TelMV mutants could systemically infect passion fruit plants without noticeable symptoms. Pre‐inoculation of one of these attenuated mutants confers efficient protection against subsequent infection by severe TelMV strain. Moreover, we demonstrated that the HC‐Pros harbored by the attenuated mutants exhibit reduced RNA silencing suppression activity in Nicotiana benthamiana leaves. CONCLUSION: The attenuated TelMV mutants developed in this study that are suitable for cross protection offer a practical, powerful tool to fight against TelMV for sustainable passion fruit production. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Introduction: Cytomegaloviruses (CMVs) extensively reorganize the membrane system of the cell and establish a new structure as large as the cell nucleus called the assembly compartment (AC). Our previous studies on murine CMV (MCMV)- infected fibroblasts indicated that the inner part of the AC contains rearranged early endosomes, recycling endosomes, endosomal recycling compartments and trans-Golgi membrane structures that are extensively tubulated, including the expansion and retention of tubular Rab10 elements. An essential process that initiates Rab10-associated tubulation is cargo sorting and retrieval mediated by SNX27, Retromer, and ESCPE-1 (endosomal SNX-BAR sorting complex for promoting exit 1) complexes. Objective: The aim of this study was to investigate the role of SNX27:Retromer: ESCPE-1 complexes in the biogenesis of pre-AC in MCMV-infected cells and subsequently their role in secondary envelopment and release of infectious virions. Results: Here we show that SNX27:Retromer:ESCPE1-mediated tubulation is essential for the establishment of a Rab10-decorated subset of membranes within the pre-AC, a function that requires an intact F3 subdomain of the SNX27 FERM domain. Suppression of SNX27-mediated functions resulted in an almost tenfold decrease in the release of infectious virions. However, these effects cannot be directly linked to the contribution of SNX27:Retromer:ESCPE-1-dependent tubulation to the secondary envelopment, as suppression of these components, including the F3-FERM domain, led to a decrease in MCMV protein expression and inhibited the progression of the replication cycle. Conclusion: This study demonstrates a novel and important function of membrane tubulation within the pre-AC associated with the control of viral protein expression. [ABSTRACT FROM AUTHOR]

Usutu virus (USUV) and West Nile virus (WNV) are two closely related emerging mosquito-borne flaviviruses. Their natural hosts are wild birds, but they can also cause severe neurological disorders in humans. Both viruses are efficiently suppressed by type I interferon (IFN), which interferes with viral replication, dissemination, pathogenesis and transmission. Here, we show that the replication of USUV and WNV are inhibited through a common set of IFN–induced genes (ISGs), with the notable exception of ISG20, which USUV is resistant to. Strikingly, USUV was the only virus among all the other tested mosquito-borne flaviviruses that demonstrated resistance to the 3′–5′ exonuclease activity of ISG20. Our findings highlight that the intrinsic resistance of the USUV genome, irrespective of the presence of cellular or viral proteins or protective post-transcriptional modifications, relies on a unique sequence present in its 3′ untranslated region. Importantly, this genomic region alone can confer ISG20 resistance to a susceptible flavivirus, without compromising its infectivity, suggesting that it could be acquired by other flaviviruses. This study provides new insights into the strategy employed by emerging flaviviruses to overcome host defense mechanisms. Here, the authors identify in the 3′ untranslated region of the genome of Usutu virus, an emerging mosquito-borne flavivirus, a sequence that confers a unique capacity to resist degradation by the ISG20 exonuclease. [ABSTRACT FROM AUTHOR]

ATG10S is a newly discovered subtype of the autophagy protein ATG10. It promotes complete macroautophagy/autophagy, degrades multiple viral proteins, and increases the expression of type III interferons. Here, we aimed to investigate the mechanism of ATG10S cooperation with IFNL1 to degrade viral proteins from different viruses. Using western blot, immunoprecipitation (IP), tandem sensor RFP-GFP-LC3B and in situ proximity ligation assays, we showed that exogenous recombinant ATG10S protein (rHsATG10S) could enter into cells through clathrin, and ATG10S combined with ATG7 with IFNL1 assistance to facilitate ATG12–ATG5 conjugation, thereby contributing to the autophagosome formation in multiple cell lines containing different virions or viral proteins. The results of DNA IP and luciferase assays also showed that ATG10S was able to directly bind to a core motif (CAAGGG) within a binding site of transcription factor ZNF460 on the IFNL1 promoter, by which IFNL1 transcription was activated. These results clarified that ATG10S promoted autophagosome formation with the assistance of IFNL1 to ensure autophagy flux and autophagic degradation of multiple viral proteins and that ATG10S could also act as a novel transcription factor to promote IFNL1 gene expression. Importantly, this study further explored the antiviral mechanism of ATG10S interaction with type III interferon and provided a theoretical basis for the development of ATG10S into a new broad-spectrum antiviral protein drug. Abbreviation: ATG: autophagy related; ATG10S: the shorter isoform of autophagy-related 10; CC50: half cytotoxicity concentration; CCV: clathrin-coated transport vesicle; CLTC: clathrin heavy chain; CM: core motif; co-IP: co-immunoprecipitation; CPZ: chlorpromazine; ER: endoplasmic reticulum; HCV: hepatitis C virus; HBV: hepatitis B virus; HsCoV-OC43: Human coronavirus OC43; IFN: interferon; PLA: proximity ligation assay; rHsATG10S: recombinant human ATG10S protein; RLU: relative light unit; SQSTM1: sequestosome 1; ZNF: zinc finger protein [ABSTRACT FROM AUTHOR]

The incessant arms race between viruses and hosts has led to numerous evolutionary innovations that shape life’s evolution. During this process, the interactions between viral receptors and viruses have garnered significant interest since viral receptors are cell surface proteins exploited by viruses to initiate infection. Our study sheds light on the arms race between the MDA5 receptor and 5’ppp-RNA virus in a lower vertebrate fish, Miichthys miiuy. Firstly, the frequent and independent loss events of RIG-I in vertebrates prompted us to search for alternative immune substitutes, with homology-dependent genetic compensation response (HDGCR) being the main pathway. Our further analysis suggested that MDA5 of M. miiuy and Gallus gallus, the homolog of RIG-I, can replace RIG-I in recognizing 5’ppp-RNA virus, which may lead to redundancy of RIG-I and loss from the species genome during evolution. Secondly, as an adversarial strategy, 5’ppp-RNA SCRV can utilize the m6A methylation mechanism to degrade MDA5 and weaken its antiviral immune ability, thus promoting its own replication and immune evasion. In summary, our study provides a snapshot into the interaction and coevolution between vertebrate and virus, offering valuable perspectives on the ecological and evolutionary factors that contribute to the diversity of the immune system. [ABSTRACT FROM AUTHOR]

Coxsackievirus B (CVB) is the major causative pathogen for severe diseases such as viral myocarditis, meningitis, and pancreatitis. There is no effective antiviral therapy currently available for CVB infection primarily due to that the pathogenesis of CVB has not been completely understood. Viruses are obligate intracellular pathogens which subvert cellular processes to ensure viral replication. Dysregulation of ubiquitination has been implicated in CVB infection. However, how ubiquitination is involved in CVB infection remains unclear. Here we found that the 3D protein of CVB3, the RNA-dependent RNA polymerase, was modified at by K48-linked polyubiquitination which promoted its degradation through proteasome. Proteomic analysis showed that the E3 ligase TRIM56 was upregulated in CVB3-infected cells, while the majority of TRIMs remained unchanged. Pull-down and immunoprecipitation analyses showed that TRIM56 interacted with CVB3 3D. Immunofluorescence observation showed that viral 3D protein was colocalized with TRIM56. TRIM56 overexpression resulted in enhanced ubiquitination of CVB3 3D and decreased virus yield. Moreover, TRIM56 was cleaved by viral 3C protease in CVB3-infected cells. Taken together, this study demonstrated that TRIM56 mediates the ubiquitination and proteasomal degradation of the CVB3 3D protein. These findings demonstrate that TRIM56 is an intrinsic cellular restriction factor against CVB infection, and enhancing viral protein degradation could be a potential strategy to control CVB infection. Author summary: Coxsackievirus B (CVB) is the major causative agent for a range of human diseases. As small infectious agent with a simple genome, CVB relies on the subversion of host processes to replicate. On the other hand, host cells contain antiviral strategies to restrict CVB replication. The present study aims to address the role played by a cellular protein termed TRIM56 in the infection of CVB3, a representative serotype of CVB, which is the causative agent of inflammatory heart disease. Our study found that viral RNA replicase 3D harbors a type of covalent modification termed polyubiquitination, resulting in the accelerated degradation of this viral replicase. We show that TRIM56 is the cellular enzyme which promotes the polyubiquitination of viral 3D protein. We demonstrated that when TRIM56 was overexpressed in the cell, the production of viral particles was significantly reduced. Moreover, we found that the protease 3C of CVB3 counteracts the antiviral effect of TRIM56 through its protease activity. Together, these data demonstrate that TRIM56 is an intrinsic cellular restriction factor against CVB infection. Enhancing viral protein degradation could be a potential strategy to control CVB infection. [ABSTRACT FROM AUTHOR]