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 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 signal-regulated kinases 1 and 2 (ERK1/2) 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 ERK1/2 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.

尽管丙型肝炎病毒（hepatitis C virus, HCV）感染是一种正在蔓延的全球性传染病，可引发严重肝脏疾病，但HCV致病的分子机制仍未完全阐明。HCV的NS5A非结构蛋白包含多个富脯氨酸序列，与细胞信号分子中存在的Src同源结构域3（Src homology 3, SH3）结合位点特征一致。本研究证实，NS5A可特异性结合生长因子受体结合蛋白2（growth factor receptor-bound protein 2, Grb2）接头蛋白。对表达NS5A的重组痘苗病毒（vaccinia virus, VV）感染的HeLa S3细胞进行抗Grb2免疫复合物的免疫印迹分析，结果显示NS5A与Grb2在活体内存在相互作用。Grb2的N端SH3结构域发生失活性点突变后，其与NS5A的结合能力显著减弱，而C端SH3结构域突变则无此效应。但若两个SH3结构域均引入相同突变，则Grb2与NS5A的结合被完全阻断，提示两个SH3结构域可通过协同方式与NS5A结合。进一步对NS5A进行突变分析，确定其SH3结合区域为一段在HCV各基因型中高度保守的富脯氨酸基序。值得注意的是，在表达NS5A的重组VV感染的HeLa S3细胞中，细胞外调节蛋白激酶1和2（extracellular signal-regulated kinases 1 and 2, ERK1/2）的磷酸化受到抑制，而空载重组VV感染的对照组细胞则无此现象。此外，稳定表达NS5A的HeLa细胞对外源性表皮生长因子诱导的ERK1/2磷酸化产生抵抗。同时，表皮生长因子刺激可诱导该细胞中NS5A与Grb2的结合。因此，NS5A可通过选择性靶向接头蛋白Grb2，干扰其介导的信号通路。本研究结果揭示了一种可拦截细胞信号通路的病毒蛋白，其对HCV致病机制具有潜在的研究价值。