Institute of Basic Medical Science, College of Medicine, National Cheng Kung University , Tainan 70401, Taiwan, Republic of China.
J Proteome Res. 2011 Oct 7;10(10):4522-34. doi: 10.1021/pr200338d. Epub 2011 Aug 24.
The hepatitis C virus core protein (HCVc) forms the viral nucleocapsid and is involved in viral persistence and pathogenesis, possibly by interacting with host factors to modulate viral replication and cellular functions. Here, we identified 36 cellular protein candidates by one-dimensional SDS-PAGE and LC-MS/MS-based proteomics after affinity purification with HCVc174, a matured form of HCVc from HCV-1b genotype, tagged with biotin and calmodulin-binding peptide/protein A at N- and C-termini, respectively. By pull-down and confocal imaging techniques, we confirmed that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1), nuclear factor 45 (NF45), and C14orf166 are novel HCVc174-interacting host proteins, known to participate in mRNA metabolism, gene regulation, and microtubule organization, respectively. Unlike the other 2 proteins, NF45 interacted with HCVc174 in an RNA-dependent manner. These 3 proteins colocalized with ectopic HCVc-1b in both the cytoplasm and nucleus, which demonstrated their spatial interaction with naturally translocated HCVc174 after HCVc biogenesis. Such colocalization, however, shifted to the cytoplasm in cells with replicating virus of 1b or 2a genotype, indicating that active viral replication confined these interacting proteins in the cytoplasm. Collectively, our findings suggest that spatial interactions of hnRNPH1, NF45, and C14orf166 with HCVc174 likely modulate HCV or cellular functions during acute and chronic HCV infection.
丙型肝炎病毒核心蛋白 (HCVc) 形成病毒核衣壳，参与病毒持续存在和发病机制，可能通过与宿主因子相互作用来调节病毒复制和细胞功能。在这里，我们通过亲和纯化用 HCVc174（HCVc 的成熟形式，来自 HCV-1b 基因型，分别在 N 端和 C 端标记生物素和钙调蛋白结合肽/蛋白 A）进行一维 SDS-PAGE 和基于 LC-MS/MS 的蛋白质组学鉴定了 36 种细胞蛋白候选物。通过下拉和共聚焦成像技术，我们证实异质核核糖核蛋白 H1 (hnRNPH1)、核因子 45 (NF45) 和 C14orf166 是 HCVc174 的新型宿主蛋白相互作用蛋白，分别参与 mRNA 代谢、基因调控和微管组织。与其他 2 种蛋白不同，NF45 以 RNA 依赖的方式与 HCVc174 相互作用。这 3 种蛋白质与细胞质和核内异位 HCVc-1b 共定位，这表明它们在 HCVc 生物发生后与天然易位的 HCVc174 具有空间相互作用。然而，在复制 1b 或 2a 基因型病毒的细胞中，这种共定位转移到细胞质中，表明活跃的病毒复制将这些相互作用的蛋白质局限在细胞质中。总之，我们的研究结果表明，hnRNPH1、NF45 和 C14orf166 与 HCVc174 的空间相互作用可能在急性和慢性 HCV 感染期间调节 HCV 或细胞功能。
