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
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
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DOI：
10.1073/pnas.96.10.5533
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发表时间：
1999-05-11
影响因子：
11.1
通讯作者：
Katze, MG
中科院分区：
文献类型：
--
作者：
Tan, SL;Nakao, H;Katze, MG
尽管丙型肝炎病毒 (HCV) 感染是一种新出现的全球流行病，会导致严重的肝脏疾病，但 HCV 发病机制的分子机制仍不清楚。 HCV 的 NS5A 非结构蛋白含有几个富含脯氨酸的序列，与细胞信号分子中发现的 Src 同源 (SH) 3 结合位点一致。在这里，我们证明 NS5A 特异性结合生长因子受体结合蛋白 2 (Grb2) 接头蛋白。对感染表达 NS5A 的重组牛痘病毒 (VV) 的 HeLa S3 细胞衍生的抗 Grb2 免疫复合物的免疫印迹分析揭示了 NS5A 和 Grb2 之间在体内的相互作用。 Grb2 N 端 SB3 结构域（而非 C 端 SH3 结构域）的失活点突变显示出与 NS5A 的结合显着减弱。然而，两个 SH3 区域中的相同突变完全消除了 Grb2 与 NS5A 的结合，这意味着两个 SH3 结构域以协作方式与 NS5A 结合。此外，NS5A 的突变分析将 SH3 结合区分配给在 HCV 中高度保守的富含脯氨酸的基序。重要的是，在感染表达重组 VV 的 NS5A 的 HeLa S3 细胞中，细胞外信号调节激酶 1 和 2 (ERK1/2) 的磷酸化受到抑制，但重组 VV 对照则不受抑制。此外，稳定表达 NS5A 的 HeLa 细胞对外源表皮生长因子诱导的 ERK1/2 磷酸化具有抵抗力。此外，这些细胞中 NS5A 与 Grb2 的偶联是由表皮生长因子刺激诱导的。因此，NS5A 可能通过选择性靶向接头来扰乱 Grb2 介导的信号通路。这些发现强调了细胞信号传导的病毒拦截器对 HCV 发病机制的潜在影响。
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 SB3 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.