Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 308 Althouse Laboratory, University Park, PA 16802, USA.
Virology. 2010 Mar 1;398(1):21-37. doi: 10.1016/j.virol.2009.11.034. Epub 2009 Dec 16.
During infection, hepatitis C virus (HCV) NS4B protein remodels host membranes to form HCV replication complexes (RC) which appear as foci under fluorescence microscopy (FM). To understand the role of Rab proteins in forming NS4B foci, cells expressing the HCV replicon were examined biochemically and via FM. First, we show that an isolated NS4B-bound subcellular fraction is competent for HCV RNA synthesis. Further, this fraction is differentially enriched in Rab1, 2, 5, 6 and 7. However, when examined via FM, NS4B foci appear to be selectively associated with Rab5 and Rab7 proteins. Additionally, dominant negative (DN) Rab6 expression impairs Rab5 recruitment into NS4B foci. Further, silencing of Rab5 or Rab7 resulted in a significant decrease in HCV genome replication. Finally, expression of DN Rab5 or Rab7 led to a reticular NS4B subcellular distribution, suggesting that endocytic proteins Rab5 and Rab7, but not Rab11, may facilitate NS4B foci formation.
在感染过程中，丙型肝炎病毒 (HCV) NS4B 蛋白重塑宿主膜，形成 HCV 复制复合物 (RC)，在荧光显微镜 (FM) 下呈现焦点。为了了解 Rab 蛋白在形成 NS4B 焦点中的作用，通过生化和 FM 检查表达 HCV 复制子的细胞。首先，我们表明，分离的 NS4B 结合亚细胞级分具有 HCV RNA 合成的能力。此外，该级分在 Rab1、2、5、6 和 7 中差异富集。然而，通过 FM 检查时，NS4B 焦点似乎与 Rab5 和 Rab7 蛋白选择性相关。此外，显性负性 (DN) Rab6 表达会损害 Rab5 招募到 NS4B 焦点中。此外，Rab5 或 Rab7 的沉默导致 HCV 基因组复制显著减少。最后，DN Rab5 或 Rab7 的表达导致 NS4B 亚细胞分布呈网状，表明内吞蛋白 Rab5 和 Rab7，但不是 Rab11，可能有助于 NS4B 焦点的形成。
