Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405;
Mol Cell Proteomics. 2014 Jan;13(1):84-92. doi: 10.1074/mcp.M113.031682. Epub 2013 Oct 10.
Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.
茎环结构 I（SL1）位于丙型肝炎病毒（HCV）基因组的 5'非翻译区，起始与 miR-122 结合，miR-122 是 HCV 复制所必需的 microRNA。然而，与 SL1 结合的蛋白质仍然难以捉摸。在这项研究中，我们使用了一种人类蛋白质组微阵列，其中包含约 17000 种全长的单独纯化的人类蛋白质，并鉴定出 313 种识别 HCV SL1 的蛋白质。鉴定出的 83 种蛋白质被注释为肝脏表达蛋白，其中 12 种已知与肝炎病毒有关。用 siRNA 诱导 12 个候选基因中的 8 个基因沉默，导致 HCV 复制效率至少降低 25%。特别是，异质性核核糖核蛋白 K（hnRNP K）的敲低以浓度依赖的方式降低 HCV 复制。紫外线交联实验也表明，hnRNP K 在 pre-mRNA 加工和运输中发挥作用，与 HCV SL1 结合最强。我们观察到，hnRNP K 作为一种核蛋白，在 HCV 表达细胞中重新定位到细胞质中。从稳定表达 HCV 复制子的 Huh7.5 细胞中免疫沉淀 hnRNP K 导致 SL1 共同免疫沉淀。这项工作鉴定出一种细胞蛋白，它可能在 HCV RNA 基因表达和代谢的调节中发挥重要作用。
