NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling

Although hepatitis C virus (HCV) infection is an emerging global epidemic c ausing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several pro line-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifical ly bound to growth Factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cell s infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutatio n in the N-terminal SB3 domain, but not in the C-terminal SH3 domain, of Gr b2 displayed significant diminished binding to NS5A, However, the same muta tion in both SH3 regions completely abrogated Grb2 binding to NS5A, implyin g that the two SH3 domains bind in cooperative fashion to NS5A, Further, mu tational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes, Importantly, phosphory lation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was inhib ited in HeLa S3 cells infected with NS5A expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A wer e refractory to ERK1/2, phosphorylation induced by exogenous epidermal grow th factor. Moreover, the coupling of NS5A to Grb2 in these cells was induce d by epidermal growth factor stimulation. Therefore, NS5A may function to p erturb Grb2-mediated signaling pathways by selectively targeting the adapto r. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.