Chemokines and inflammatory cytokines are key regulators of immunity and inflammation during viral infections. Hepatitis C virus (HCV) is a hepatotropic RNA virus frequently associated with chronic liver inflammation and hepatocellular carcinoma. Intrahepatic levels of chemokines and cytokines are elevated in chronic HCV infections, but the underlying mechanisms remain unclear. We find that Toll like receptor-3 (TLR3) senses HCV infection in cultured hepatoma cells, leading to NF-κB activation and the production of numerous chemokines and inflammatory cytokines, such as RANTES, MIP-1α, MIP-1β, IP-10 and IL-6. The chemokine/cytokine induction occurred late in HCV infection and was abrogated when HCV was UV-inactivated prior to infection, indicating a dependence on cellular recognition of HCV replication products. Gel shift and chromatin immunoprecipitation assays revealed that NF-κB plays a pivotal role in HCV-induced chemokine/cytokine transcription. Mutations specifically disrupting the dsRNA binding activity of TLR3 ablated the chemokine/cytokine response to HCV infection, indicating that HCV dsRNA was the pathogen associated molecular pattern triggering TLR3 signaling. In vitro synthesized HCV dsRNAs with a minimal length of ~80-100 bp activated TLR3-dependent chemokine expression, regardless of the genome position from which they derive. In contrast, HCV ssRNAs, including those derived from the structured 3′NTR highly potent for RIG-I activation, failed to do so. Moreover, robust production of chemokines and inflammatory cytokines was also observed in primary human hepatocytes following stimulation with extracellular poly-I:C, a TLR3 ligand. Conclusion Our data suggest that TLR3-mediated chemokine and inflammatory cytokine responses may play an important role in host immune responses to HCV and the pathogenesis of HCV-associated liver diseases.

Background:The role of TRIM56 in innate antiviral immunity is unclear. Results: Knockdown of TRIM56 impairs TLR3-dependent IRF3 activation, interferon-stimulated gene expression and induction of antiviral responses, while overexpression of TRIM56 enhances TLR3-mediated innate immune responses. Conclusion: TRIM56 interacts with TRIF and promotes TLR3 signaling. Significance: This study identifies TRIM56 as an essential component of the TLR3 antiviral signaling pathway.

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. IMPORTANCEWe 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.

Accumulating data suggest that tripartite-motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus, and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Here, we show that TRIM56 puts a check on replication of influenza A and B viruses in cell culture but does not inhibit Sendai virus or human metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza virus activity was independent of the E3 ligase activity, B-box, or coiled-coil domain. Rather, deletion of a 63-residue-long C-terminal-tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Together, these data reveal a novel antiviral activity of TRIM56 against influenza A and B viruses and provide insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. IMPORTANCE Options to treat influenza are limited, and drug-resistant influenza virus strains can emerge through minor genetic changes. Understanding novel virus-host interactions that alter influenza virus fitness may reveal new targets/approaches for therapeutic interventions.We show here that TRIM56, a tripartite-motif protein, is an intrinsic host restriction factor of influenza A and B viruses. Unlike its antiviral actions against positive-strand RNA viruses, the anti-influenza virus activity of TRIM56 was independent of the E3 ligase activity. Rather, expression of a short segment within the very C-terminal tail of TRIM56 inhibited the replication of influenza viruses as effectively as that of full-length TRIM56 by specifically targeting viral RNA synthesis. These data reveal the remarkable multifaceted activity of TRIM56, which has developed multiple domains to inhibit multiple viral families. They also raise the possibility of developing a broad-spectrum, TRIM56-based antiviral approach for addition to influenza prophylaxis and/or control strategies.

Infection by Zika virus (ZIKV) is linked to microcephaly and other neurological disorders, posing a significant health threat. Innate immunity is the first line of defense against invading pathogens, but relatively little is understood regarding host intrinsic mechanisms that guard against ZIKV. Here, we show that host tripartite motif-containing protein 56 (TRIM56) poses a barrier to ZIKV infection in cells of neural, epithelial and fibroblast origins. Overexpression of TRIM56, but not an E3 ligase-dead mutant or one lacking a short C-terminal portion, inhibited ZIKV RNA replication. Conversely, depletion of TRIM56 increased viral RNA levels. Although the C-terminal region of TRIM56 bears sequence homology to NHL repeat of TRIM-NHL proteins that regulate miRNA activity, knockout of Dicer, which abolishes production of miRNAs, had no demonstrable effect on ZIKV restriction imposed by TRIM56. Rather, we found that TRIM56 is an RNA-binding protein that associates with ZIKV RNA in infected cells. Moreover, a recombinant TRIM56 fragment comprising the C-terminal 392 residues captured ZIKV RNA in cell-free reactions, indicative of direct interaction. Remarkably, deletion of a short C-terminal tail portion abrogated the TRIM56-ZIKV RNA interaction, concomitant with a loss in antiviral activity. Altogether, our study reveals TRIM56 is an RNA binding protein that acts as a ZIKV restriction factor and provides new insights into the antiviral mechanism by which this E3 ligase tackles flavivirus infections.

Toll-like receptor-3 (TLR3) senses double-stranded RNA intermediates produced during hepatitis C virus (HCV) replication, leading to activation of interferon regulatory factor-3 (IRF3) and NF-B and subsequent antiviral and proinflammatory responses. Yet, how this TLR3-dependent pathway operates in hepatocytes is unclear. Upon fractionating cultured hepatocytes into various cellular organelles, we observed that TLR3 predominantly resides in endolysosomes of hepatocytes. To determine the critical regulators of TLR3 signaling in response to HCV infection in human hepatocytes, we isolated endolysosome fractions from mock-infected and HCV-infected hepatoma Huh7.5 cells that had been reconstituted for TLR3 expression, separated these fractions on two-dimensional gels, and identified up-regulated/down-regulated proteins by mass spectrometry. Approximately a dozen of cellular proteins were found to be differentially expressed in endolysosome fractions following HCV infection. Of these, expression of several molecular chaperone proteins was elevated. Knockdown of one of these chaperones, glucose-regulated protein 78 kDa (GRP78), compromised TLR3-dependent induction of interferonstimulated genes and chemokines following HCV infection or poly(I:C) stimulation in cultured hepatocytes. Consistent with this finding, GRP78 depletion impaired TLR3-mediated establishment of an antiviral state. Mechanistically, although TLR3 trafficking to endolysosomes was not affected, phosphorylated IRF3 diminished faster following GRP78 knockdown. Remarkably, GRP78 transcript was significantly up-regulated in liver biopsies of chronic hepatitis C patients as compared with normal liver tissues. Moreover, the GRP78 expression level correlated with that of RANTES (regulated upon activation, normal T-cell expressed and secreted) and CXCL10, two inflammatory chemokines most frequently elevated in HCV-infected liver. Altogether, our data suggest that GRP78 contributes to TLR3-mediated, IRF3-dependent innate immune response to HCV in hepatocytes. Infection with the hepatitis C virus (HCV)2 is associated with relatively robust transcriptional induction of interferon (IFN)-stimulated genes (ISGs) and inflammatory mediators in the liver (1, 2). This innate immune response provides the first wave of cellular defense against HCV and orchestrates the development of anti-HCV adaptive immunity, which determines infection outcome (3, 4). In support of this concept, genetic polymorphisms around the IFNL3/IFNL4 gene locus and ISG expression status prior to treatment are associated with spontaneous HCV clearance and/or response to IFN-based therapies (5-9).During viral infections, pattern recognition receptors such as the retinoic acid-inducible gene-I (RIG-I)-like receptors (RIG-I and MDA5) and Toll-like receptors (TLRs) are innate immune sensors that detect viral components as non-self materials and initiate signaling cascades, leading to activation of the latent transcription factors, IRF3 and NF-B, which culminates in expression of IFNs, ISGs, cytokines, an...

The activation of interferon (IFN)-regulatory factor-3 (IRF3), characterized by phosphorylation and nuclear translocation of the latent transcription factor, is central to initiating innate antiviral responses. Whereas much has been learned about the upstream pathways and signaling mechanisms leading to IRF3 activation, how activated IRF3 operates in the nucleus to control transcription of IFNs remains obscure. Here we identify EAP30 (a.k.a, SNF8/VPS22), an endosomal sorting complex required for transport (ESCRT)-II subunit, as an essential factor controlling IRF3-dependent antiviral defense. Depletion of EAP30, but not other ESCRT-II subunits, compromised IRF3-dependent induction of type I and III IFNs, IFN-stimulated genes (ISGs) and chemokines by double-stranded RNA or viruses. EAP30, however, was dispensable for the induction of inflammatory mediators of strict NF-κB target. Significantly, knockdown of EAP30 also impaired the establishment of an antiviral state against vesicular stomatitis virus and hepatitis C virus, which are of distinct viral families. Mechanistically, EAP30 was not required for IRF3 activation but rather acted at a downstream step. Specifically, a fraction of EAP30 localized within the nucleus, where it formed a complex with IRF3 and its transcriptional co-activator, CREB-binding protein (CBP), in a virus-inducible manner. These interactions promoted IRF3 binding to target gene promoters such as IFN-β, IFN-λ1 and ISG56. Together, our data describe an unappreciated role for EAP30 in IRF3-dependent innate antiviral response in the nucleus.

Accumulating data suggest that tripartite motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Herein, we show TRIM56 puts a check on replication of influenza A and B viruses in cell culture, but does not inhibit Sendai virus or human Metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza activity was independent of the E3 ligase activity, B-box, or coiled-coil domains. Rather, deletion of a 63-residue long, C-terminal tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Consistent with this, a fraction of TRIM56 was re-distributed to the nucleus after IAV infection. Together, these data reveal novel antiviral activity of TRIM56 against influenza A and B viruses and provide novel insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. These findings raise the possibility that small molecules mimicking the C-terminal portion of TRIM56 could be developed into broad influenza antivirals.