The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity.

The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1T946) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1T946 exhibited heightened basal and ligand-triggered production of type I interferons. Consistent with those findings, mice with a knock-in mutation encoding IFIH1T946 displayed enhanced basal expression of type I interferons, survived a lethal viral challenge and exhibited increased penetrance in autoimmune models, including a combinatorial effect with other risk variants. Furthermore, IFIH1T946 mice manifested an embryonic survival defect consistent with enhanced responsiveness to RNA self ligands. Together our data support a model wherein the production of type I interferons driven by an autoimmune risk variant and triggered by ligand functions to protect against viral challenge, which probably accounts for its selection within human populations but provides this advantage at the cost of modestly promoting the risk of autoimmunity.

SIDT2 Transports Extracellular dsRNA into the Cytoplasm for Innate Immune Recognition.

Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.

OASL1 inhibits translation of the type I interferon-regulating transcription factor IRF7.

The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5' untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1-/- mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.

Pellino3 targets the IRF7 pathway and facilitates autoregulation of TLR3- and viral-induced expression of type I interferons.

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

SUMOylation of DDX39A Alters Binding and Export of Antiviral Transcripts to Control Innate Immunity.

The RNA helicase DDX39A plays an important role in the RNA splicing/export process. In our study, human DDX39A facilitated RNA virus escape from innate immunity to promote virus proliferation by trapping TRAF3, TRAF6, and MAVS mRNAs in the HEK293T cell nucleus. DDX39A was a target for SUMOylation. SUMO1, 2, and 3 modifications were found on immunoprecipitated DDX39A. However, only the SUMO1 modification decreased in vesicular stomatitis virus-infected HEK293T cells. Further studies have found that viral infection reduced SUMO1 modification of DDX39A and enhanced its ability to bind innate immunity-associated mRNAs by regulating the abundance of RanBP2 with SUMO1 E3 ligase activity. RanBP2 acted as an E3 SUMO ligase of DDX39A, which enhanced SUMO1 modification of DDX39A and attenuated its ability to bind RNA. This work described that specific mRNAs encoding antiviral signaling components were bound and sequestered in the nucleus by DDX39A to limit their expression, which proposed a new protein SUMOylation model to regulate innate immunity in viral infection.

Mastomys natalensis is a possible natural rodent reservoir for encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) infects a wide range of hosts and can cause encephalitis, myocarditis, reproductive disorders and diabetes mellitus in selected mammalian species. As for humans, EMCV infection seems to occur by the contact with animals and can cause febrile illnesses in some infected patients. Here we isolated EMCV strain ZM12/14 from a natal multimammate mouse (Mastomys natalensis: M. natalensis) in Zambia. Pairwise sequence similarity of the ZM12/14 P1 region consisting of antigenic capsid proteins showed the highest similarity of nucleotide (80.7 %) and amino acid (96.2%) sequence with EMCV serotype 1 (EMCV-1). Phylogenetic analysis revealed that ZM12/14 clustered into EMCV-1 at the P1 and P3 regions but segregated from known EMCV strains at the P2 region, suggesting a unique evolutionary history. Reverse transcription PCR (RT-PCR) screening and neutralizing antibody assays for EMCV were performed using collected tissues and serum from various rodents (n=179) captured in different areas in Zambia. We detected the EMCV genome in 19 M. natalensis (19/179=10.6 %) and neutralizing antibody for EMCV in 33 M. natalensis (33/179=18.4 %). However, we did not detect either the genome or neutralizing antibody in other rodent species. High neutralizing antibody litres (≧320) were observed in both RT-PCR-negative and -positive animals. Inoculation of ZM12/14 caused asymptomatic persistent infection in BALB/c mice with high antibody titres and high viral loads in some organs, consistent with the above epidemiological results. This study is the first report of the isolation of EMCV in Zambia, suggesting that M. natalensis may play a role as a natural reservoir of infection.

Role of pattern recognition receptors and interferon-beta in protecting bat cell lines from encephalomyocarditis virus and Japanese encephalitis virus infection.

Bats are potential natural hosts of Encephalomyocarditis virus (EMCV) and Japanese encephalitis virus (JEV). Bats appear to have some unique features in their innate immune system that inhibit viral replication causing limited clinical symptoms, and thus, contributing to the virus spill over to humans. Here, kidney epithelial cell lines derived from four bat species (Pteropus dasymallus, Rousettus leschenaultii, Rhinolophus ferrumequinum, and Miniopterus fuliginosus) and two non-bat species (Homo sapiens and Mesocricetus auratus) were infected with EMCV and JEV. The replication of EMCV and JEV was lower in the bat cell lines derived from R. leschenaultii, R. ferrumequinum, and M. fuliginosus with a higher expression level of pattern recognition receptors (PRRs) (TLR3, RIG-I, and MDA5) and interferon-beta (IFN-ß) than that in the non-bat cell lines and a bat cell line derived from P. dasymallus. The knockdown of TLR3, RIG-I, and MDA5 in Rhinolophus bat cell line using antisense RNA oligonucleotide led to decrease IFN-ß expression and increased viral replication. These results suggest that TLR3, RIG-I, and MDA5 are important for antiviral response against EMCV and JEV in Rhinolophus bats.

DHX29 functions as an RNA co-sensor for MDA5-mediated EMCV-specific antiviral immunity.

Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is an RNA helicase required for the translation of 5' structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5' structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity.

PKR Transduces MDA5-Dependent Signals for Type I IFN Induction.

Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVΔE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluΔNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2α, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVΔE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism.

Construction, expression, and characterization of a single-chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, applying gene splicing by overlap extension PCR (SOE-PCR), we describe a simple method for producing single-chain variable fragment (scFv) antibody against EMCV that configurates in the orientation of VH-(GGGGS)4 -VL. DNA template was resverse transcribed by total RNA that derived from hyperimmunized antibody positive mice spleen after inoculation inactivated EMCV-PV21 as antigen. Using the degenerate primers designed for the variable regions of IgG of murine antibody, the 417 bp of gene encoding VH-linker (VHL) and 360 bp of gene encoding linker-VL (LVL) of the anti-EMCV was individually amplified from DNA template by PCR, repectively. The 762 bp gene encoding anti-EMCV scFv was constructed by SOE-PCR when the mixed VHL and LVL genes were used as the template. The amplified gene subcloned into pGEX-6P1 to yield pGEX-6P1/EMCV-scFv. Recombinant vector transformed into the Escherichia coli BL21 (DE3) and a 53 KDa GST-scFv fusion protein was obtained by SDS-PAGE electrophoresis. Animal experiment results showed that the pretective rate of mice in group A which challenged 500 µL 104 TCID50 EMCV per mouse for 7 d post-inoculation scFv 3 d (0.5 mg purified recombinant scFv per mouse) was 91.67% (11/12). The serum anti-EMCV antibody titer in group A mice was most significantly higher than that in positive control mouse (P < 0.01), coversely the serum relative mRNA copies were significantly lower than that in positive control mouse (P < 0.05). These findings indicated that recombinant anti-EMCV scFv has remarkable anti-EMCV effect in mice.

MOV10 Provides Antiviral Activity against RNA Viruses by Enhancing RIG-I-MAVS-Independent IFN Induction.

Moloney leukemia virus 10, homolog (MOV10) is an IFN-inducible RNA helicase, associated with small RNA-induced silencing. In this article, we report that MOV10 exhibits antiviral activity, independent of its helicase function, against a number of positive- and negative-strand RNA viruses by enhancing type I IFN induction. Using a number of genome-edited knockout human cells, we show that IFN regulatory factor 3-mediated IFN induction and downstream IFN signaling through IFN receptor was necessary to inhibit virus replication by MOV10. MOV10 enhanced IFN regulatory factor 3-mediated transcription of IFN. However, this IFN induction by MOV10 was unique and independent of the known retinoic acid-inducible gene I/mitochondrial antiviral-signaling protein-mediated RNA-sensing pathway. Upon virus infection, MOV10 specifically required inhibitor of κB kinase ε, not TANK-binding kinase 1, for its antiviral activity. The important role of MOV10 in mediating antiviral signaling was further supported by the finding that viral proteases from picornavirus family specifically targeted MOV10 as a possible innate immune evasion mechanism. These results establish MOV10, an evolutionary conserved protein involved in RNA silencing, as an antiviral gene against RNA viruses that uses an retinoic acid-inducible gene I-like receptor-independent pathway to enhance IFN response.

PACT is required for MDA5-mediated immunoresponses triggered by Cardiovirus infection via interaction with LGP2.

Laboratory of genetics and physiology 2 (LGP2) and melanoma differentiation-associated gene 5 (MDA5) cooperatively detect viral RNA in the cytoplasm of Cardiovirus-infected cells and activate innate immune responses. Here, we evaluated whether the double-stranded RNA-binding protein PACT plays a role in this anti-viral response to further elucidate the mechanism. Immunoprecipitation experiments demonstrated that PACT interacts with LGP2 and that this interaction is enhanced by encephalomyocarditis virus (EMCV) infection. In vitro interaction analyses using purified recombinant proteins confirmed that the single-stranded Theiler's murine encephalitis virus genome enhanced the interaction between LGP2 and PACT. Small interfering RNA knockdown experiments further indicated that PACT is required for Cardiovirus-triggered interferon responses. To support this functional interaction with LGP2, overexpressed PACT was shown to enhance EMCV-triggered interferon promoter activity only when LGP2 and MDA5 were co-expressed but not when MDA5 is expressed alone. Together, our findings indicate a possible role of PACT in regulating the Cardiovirus-triggered immune responses mediated by MDA5 and LGP2, which opens the door to novel therapeutic strategies in interferon-related autoimmune diseases and cancer.

Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression.

The role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lpro and 3Cpro are viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses, including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasome, lysosome, and caspase pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl-terminal amino acids 105 to 114 and amino acids 135 to 144 of 2B were essential for the reduction of RIG-I, while residues 105 to 114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein. IMPORTANCE: This study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasome, lysosome, and caspase pathways were not involved in this process. This study provides new insight into the immune evasion mediated by FMDV and identifies 2B as an antagonistic factor for FMDV to evade the antiviral response.

Transcriptional profiling of host cell responses to encephalomyocarditis virus (EMCV).

BACKGROUD: Encephalomyocarditis virus (EMCV) has been discovered on pig farms worldwide and can cause myocarditis in piglets and reproductive failure in sows. However, little is known about the host transcriptional responses to infection and host-pathogen interactions. METHODS: In this study, transcription profiling was performed by Illumina RNA-Sequencing (RNA-seq) to identify EMCV induced differentially expressed genes in BHK-21 cells at serial time points (12, 24, and 30 h post infection (hpi)), using mock infected cells as control. RESULTS: We identified 237, 241, and 207 differentially expressed genes (DEGs) respectively, majority of which were up-regulated. A large number of DEGs clustered into host defense, cellular signaling and metabolism categories. Moreover, short time series expression analysis revealed that 12 hpi was an important time point for expression change, indicating host virus resistance. CONCLUSIONS: This RNA-seq analysis provides the first data for understanding the network of virus host interactions under EMCV infection in vitro, and for identifying host components which involved in the virus infection course.

Human Cytokinome Analysis for Interferon Response.

UNLABELLED: Cytokines are a group of small secreted proteins that mediate a diverse range of immune and nonimmune responses to inflammatory and microbial stimuli. Only a few of these cytokines mount an antiviral response, including type I, II, and III interferons (IFNs). During viral infections and under inflammatory conditions, a number of cytokines and chemokines are coproduced with IFN; however, no systematic study exists on the interactions of the cytokine repertoire with the IFN response. Here, we performed the largest cytokine and chemokine screen (the human cytokinome, with >240 members) to investigate their modulation of type I and type II IFN responses in a cell line model. We evaluated the cytokine activities in both IFN-stimulated response element (ISRE) and IFN-γ activation sequence (GAS) reporter systems. Several cytokine clusters that augment either or both ISRE- and GAS-mediated responses to IFNs were derived from the screen. We identified novel modulators of IFN response-betacellulin (BTC), interleukin 11 (IL-11), and IL-17F-that caused time-dependent induction of the IFN response. The ability to induce endogenous IFN-ß and IFN-stimulated genes varies among these cytokines and was largely dependent on Stat1, as assessed by Stat1 mutant fibroblasts. Certain cytokines appear to augment the IFN-ß response through the NF-κB pathway. The novel IFN-like cytokines augmented the antiviral activity of IFN-α against several RNA viruses, including encephalomyocarditis virus, vesicular stomatitis virus, and influenza virus, in susceptible cell lines. Overall, the study represents a large-scale analysis of cytokines for enhancing the IFN response and identified cytokines capable of enhancing Stat1, IFN-induced gene expression, and antiviral activities. IMPORTANCE: Innate immunity to viruses is an early defense system to ward off viruses. One mediator is interferon (IFN), which activates a cascade of biochemical events that aim to control the virus life cycle. In our work, we examined more than 200 cytokines, soluble mediators produced within the body as a result of infection, for the ability to enhance IFN action. We identified enhanced interactions with specific IFNs and cytokines. We also revealed that betacellulin, IL-17, and IL-11 cytokines have the novel property of enhancing the antiviral action of IFN against several viruses. These results demonstrate that the human genome codes for previously unknown proteins with unrelated functions that can augment the innate immunity to viruses. Knowing these interactions not only helps our understanding of immunity to viruses and emerging diseases, but can also lead to devising possible new therapeutics by enhancing the mediator of antiviral action itself, IFN.

Platelets: crossroads of immunity and hemostasis.

In this issue of Blood, Koupenova and colleagues report that platelets express functional TOLL-like receptor 7 (TLR7) and contribute to host survival during viral infection. Through a series of experiments utilizing mice deficient for TLR7 together with adoptive transfer of wild-type platelets, Koupenova et al demonstrate that platelets specifically respond to viral analogs and intact virus, leading to platelet activation and binding to various leukocyte subsets. Perhaps most importantly, this platelet activation appears absolutely essential for host survival during infection with some viral pathogens such as encephalomyocarditis virus (EMCV).

Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis.

Viral infections have been associated with reduced platelet counts, the biological significance of which has remained elusive. Here, we show that infection with encephalomyocarditis virus (EMCV) rapidly reduces platelet count, and this response is attributed to platelet Toll-like receptor 7 (TLR7). Platelet-TLR7 stimulation mediates formation of large platelet-neutrophil aggregates, both in mouse and human blood. Intriguingly, this process results in internalization of platelet CD41-fragments by neutrophils, as assessed biochemically and visualized by microscopy, with no influence on platelet prothrombotic properties. The mechanism includes TLR7-mediated platelet granule release, translocation of P-selectin to the cell surface, and a consequent increase in platelet-neutrophil adhesion. Viral infection of platelet-depleted mice also led to increased mortality. Transfusion of wild-type, TLR7-expressing platelets into TLR7-deficient mice caused a drop in platelet count and increased survival post EMCV infection. Thus, this study identifies a new link between platelets and their response to single-stranded RNA viruses that involves activation of TLR7. Finally, platelet-TLR7 stimulation is independent of thrombosis and has implications to the host immune response and survival.

Phosphoproteomics combined with quantitative 14-3-3-affinity capture identifies SIRT1 and RAI as novel regulators of cytosolic double-stranded RNA recognition pathway.

Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses.

Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus.

UNLABELLED: The tripartite motif-containing (TRIM) proteins have emerged as a new class of host antiviral restriction factors, with several demonstrating roles in regulating innate antiviral responses. Of >70 known TRIMs, TRIM56 inhibits replication of bovine viral diarrhea virus, a ruminant pestivirus of the family Flaviviridae, but has no appreciable effect on vesicular stomatitis virus (VSV), a rhabdovirus. Yet the antiviral spectrum of TRIM56 remains undefined. In particular, how TRIM56 impacts human-pathogenic viruses is unknown. Also unclear are the molecular determinants governing the antiviral activities of TRIM56. Herein, we show that TRIM56 poses a barrier to infections by yellow fever virus (YFV), dengue virus serotype 2 (DENV2), and human coronavirus virus (HCoV) OC43 but not encephalomyocarditis virus (EMCV). Moreover, by engineering cell lines conditionally expressing various TRIM56 mutants, we demonstrated that TRIM56's antiflavivirus effects required both the E3 ligase activity that lies in the N-terminal RING domain and the integrity of its C-terminal portion, while the restriction of HCoV-OC43 relied upon the TRIM56 E3 ligase activity alone. Furthermore, TRIM56 was revealed to impair YFV and DENV2 propagation by suppressing intracellular viral RNA accumulation but to compromise HCoV-OC43 infection at a later step in the viral life cycle, suggesting that distinct TRIM56 domains accommodate differing antiviral mechanisms. Altogether, TRIM56 is a versatile antiviral host factor that confers resistance to YFV, DENV2, and HCoV-OC43 through overlapping and distinct molecular determinants. IMPORTANCE: We previously reported tripartite motif protein 56 (TRIM56) as a host restriction factor of bovine viral diarrhea virus, a ruminant pathogen. However, the impact of TRIM56 on human-pathogenic RNA viruses is unknown. Herein, we demonstrate that TRIM56 restricts two medically important flaviviruses, yellow fever virus (YFV) and dengue virus serotype 2 (DENV2), and a human coronavirus, HCoV-OC43, but not encephalomyocarditis virus, a picornavirus. Further, we show that TRIM56-mediated inhibition of HCoV-OC43 multiplication depends solely on its E3 ligase activity, whereas its restriction of YFV and DENV2 requires both the E3 ligase activity and integrity of the C-terminal portion. The differing molecular determinants appear to accommodate distinct antiviral mechanisms TRIM56 adopts to target different families of viruses; while TRIM56 curbs intracellular YFV/DENV2 RNA replication, it acts at a later step in HCoV-OC43 life cycle. These novel findings illuminate the molecular basis of the versatility and specificity of TRIM56's antiviral activities against positive-strand RNA viruses.