Shanghai Medical College, Fudan University, Shanghai 200032, China.
J Virol. 2011 Jul;85(14):6996-7004. doi: 10.1128/JVI.00013-11. Epub 2011 May 11.
We recently reported that Ras-GTPase-activating protein-binding protein 1 (G3BP1) interacts with hepatitis C virus (HCV) nonstructural protein (NS)5B and the 5' end of the HCV minus-strand RNA. In the current study we confirmed these observations using immunoprecipitation and RNA pulldown assays, suggesting that G3BP1 might be an HCV replication complex (RC) component. In replicon cells, transfected G3BP1 interacts with multiple HCV nonstructural proteins. Using immunostaining and confocal microscopy, we demonstrate that G3BP1 is colocalized with HCV RCs in replicon cells. Small interfering RNA (siRNA)-mediated knockdown of G3BP1 moderately reduces established HCV RNA replication in HCV replicon cells and dramatically reduces HCV replication-dependent colony formation and cell-culture-produced HCV (HCVcc) infection. In contrast, knockdown of G3BP2 has no effect on HCVcc infection. Transient replication experiments show that G3BP1 is involved in HCV genome amplification. Thus, G3BP1 is associated with HCV RCs and may be co-opted as a functional RC component for viral replication. These findings may facilitate understanding of the molecular mechanisms of HCV genome replication.
我们最近报道称 Ras-GTPase-activating protein-binding protein 1（G3BP1）与丙型肝炎病毒（HCV）非结构蛋白（NS）5B 和 HCV 负链 RNA 的 5'端相互作用。在本研究中，我们使用免疫沉淀和 RNA 下拉实验证实了这些观察结果，表明 G3BP1 可能是 HCV 复制复合物（RC）的组成部分。在复制子细胞中，转染的 G3BP1 与多种 HCV 非结构蛋白相互作用。通过免疫染色和共聚焦显微镜，我们证明 G3BP1 在复制子细胞中与 HCV RC 共定位。使用小干扰 RNA（siRNA）介导的 G3BP1 敲低可适度降低 HCV 复制子细胞中已建立的 HCV RNA 复制，并显著降低 HCV 复制依赖性集落形成和细胞培养产生的 HCV（HCVcc）感染。相比之下，G3BP2 的敲低对 HCVcc 感染没有影响。瞬时复制实验表明 G3BP1 参与 HCV 基因组扩增。因此，G3BP1 与 HCV RC 相关，可能被选为病毒复制的功能性 RC 成分。这些发现可能有助于理解 HCV 基因组复制的分子机制。
