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.

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...

Recently, putative viral agents responsible for human non-A to E hepatitis have been independently reported by two groups of investigators and designated GB virus C (GBV-C) and hepatitis G virus (HGV), respectively. The entire nucleotide sequences were determined for two viral genomes isolated from Japanese blood donors with GBV-C RNA. One of them (GT230) had a total genomic length of 9390 nucleotides (nt) with 5h and 3h untranslated regions of 551 and 313 nt, while the other (GT110) had genomic lengths of 9395, 281 and 315 nt, respectively. They both had a single long open reading frame, encoding 2842 amino acids (aa) in GT230 and 2933 aa in GT110. Surprisingly, they both lacked a clearly identifiable core gene, and possessed the E1/E2 gene with only four potential

Endoscopic submucosal dissection can take a long time, but is superior to conventional endoscopic mucosal resection for treating intramucosal gastric neoplasms.