Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signalregulated kinases 1 and 2 (ERK1͞2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1͞2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.

The vaccinia virus E3L gene encodes two double-stranded RNA binding proteins that promote viral growth and pathogenesis through suppression of innate immunity. To explore how E3L enables vaccinia virus to evade the interferon system, cells and mice deficient in the principal interferon-regulated antiviral enzymes, PKR and RNase L, were infected with wild-type vaccinia virus and strains of vaccinia virus from which E3L had been deleted (E3L-deleted strains). While wild-type virus was unaffected by RNase L and PKR, virus lacking E3L replicated only in the deficient cells. Nevertheless, E3L-deleted virus failed to replicate to high titers or to cause significant morbidity or mortality in triply deficient mice lacking RNase L, PKR, and Mx1. To investigate the underlying cause, we determined the effect of E3L on interferon regulatory factor 3 (IRF3), a transcription factor required for viral induction of subtypes of type I interferons. Results showed that IRF3 activation and interferon-␤ induction occurred after infections with E3L-deleted virus but not with wild-type virus. These findings demonstrate that E3L plays an essential role in the pathogenesis of vaccinia virus by blocking the interferon system at multiple levels. Furthermore, our results indicate the existence of an interferon-mediated antipoxvirus pathway that operates independently of PKR, Mx1, or the 2-5A/RNase L system.

Hepatitis C virus (HCV) sets up a persistent infection in patients that likely involves a complex virus-host interaction. We previously found that the HCV nonstructural 5A (NS5A) protein interacts with growth factor receptor-binding protein 2 (Grb2) adaptor protein and inhibits the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by epidermal growth factor (EGF). In the present study, we extended this analysis and investigated the specificity of the Grb2-NS5A interaction and whether the subversion of mitogenic signaling involves additional pathways. NS5A containing mutations within the C-terminal proline-rich motif neither bound Grb2 nor inhibited ERK1/2 activation by EGF, demonstrating that NS5A-Grb2 binding and downstream effects were due to direct interactions. Interestingly, NS5A could also form a complex with the Grb2-associated binder 1 (Gab1) protein in an EGF treatment-dependent manner. However, the NS5A-Gab1 association, which appeared indirect, was not mediated by direct NS5A-Grb2 interaction but was likely dependent on direct NS5A interaction with the p85 subunit of phosphatidylinositol 3-kinase (PI3K). The in vivo association of NS5A with p85 PI3K required the N-terminal, but not the C-terminal, region of NS5A. The downstream effects of the NS5A-p85 PI3K interaction included increased tyrosine phosphorylation of p85 PI3K in response to EGF. Consistent with this observation and the antiapoptotic properties of NS5A, we also detected enhanced tyrosine phosphorylation of the downstream AKT protein kinase and increased serine phosphorylation of BAD, a proapoptotic factor and an AKT substrate, in the presence of NS5A. These results collectively suggest a model in which NS5A interacts with Grb2 to inhibit mitogenic signaling while simultaneously promoting the PI3K-AKT cell survival pathway by interaction with p85 PI3K, which may represent a crucial step in HCV persistence and pathogenesis.Hepatitis C virus (HCV), a Flaviviridae family member, contains a positive-sense, single-stranded RNA genome that encodes about 10 mature viral structural and nonstructural (NS) proteins (41). Infecting approximately 2% of the world population, HCV is the global leading cause of chronic liver disease and has become a major public health problem in the United States (10, 11). In the majority of cases, acute infection with HCV results in persistent viral replication and establishment of a chronic infection. Chronic hepatitis C frequently leads to progressive liver disease, including liver fibrosis and cirrhosis, and is strongly associated with the onset of hepatocellular carcinoma. HCV research has been hampered by the lack of an efficient tissue culture system or an adequate animal model of HCV infection (18). As a result, the mechanisms of HCV replication, persistence, and pathogenesis remain poorly understood. Consequently, our general understanding of the impact of HCV infection on cellular signaling is far from complete or clear.HCV-host interactions have been intensely investigated despite the lack...

The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VV⌬E3L) resulted in increased phosphorylation levels of both PKR and eIF2␣. IFN-pretreated cells infected with VV in which the E3L locus was replaced with the NS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2␣ in a transient manner. We also observed an increase in activation of p38 mitogenactivated protein kinase in IFN-pretreated cells infected with VV⌬E3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the capbinding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2␣ and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VV⌬E3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.