IDENTIFICATION OF NOVEL HOST FACTORS INTERACTING WITH NS5A PROTEIN OF HCV

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 - 背景和目标 丙型肝炎病毒（HCV）是一种终身持续性感染，可引起慢性炎症性疾病，最终导致肝纤维化、肝硬化和肝细胞癌等严重的肝病并发症。 作为黄病毒科肝炎病毒属的唯一成员，这种正链极性的ssRNA病毒在感染细胞的胞质区室中复制，并引起称为“膜网”的改变的膜结构，其被认为是从粗面内质网延伸，并且与活跃复制的病毒RNA相关。尽管对这种病毒如何在细胞内建立其微环境知之甚少，但揭示HCV如何在其作为包膜病毒的病毒体生产需求的背景下处理脂质代谢及其运输正变得非常有趣。 - 方法 用亚基因组复制子RNA转染人肝癌细胞Huh-7，在复制子RNA中非结构蛋白5A（NS 5A）的C端引入亲和标签肽序列。用G418补充的培养基培养转染的细胞，以选择携带复制病毒RNA的细胞。分离得到的G418抗性细胞，发现其通过病毒复制表达亲和标记的NS 5A蛋白。通过亲和柱纯化分离病毒蛋白复合物以及它们相互作用的宿主蛋白，并通过MudPIT分析以鉴定相关的宿主因子。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. -Background and Aim Hepatitis C Virus (HCV) is known to establish a lifelong persistent infection causing patient a chronic inflammatory disease, which eventually results in severe complication of liver diseases including fibrosis, cirrhosis and hepatocellular carcinoma. As the sole member of the genus Hepacivirus of Flaviviridae family, this ssRNA virus of plus strand polarity replicates in the cytosolic compartment of infected cells and causes an altered membraneous structure called "membraneous web" which is believed to be extended from rough endoplasmic reticulum, and is associated with actively replicating viral RNA. Although little is known about how this virus establish their microenviroment inside cell, It is becoming of great interest to reveal how HCV is dealing with both lipid metabolism and its trafficking in the context of their demand for virion production as envelope virus. -Method Human hepatoma cell line, Huh-7 was transfected with subgenomic replicon RNA, into which Affinity-tag peptide sequence was introduced in the C-terminus region of Nonstructural protein 5A (NS5A). Transfected cells were cultured with G418 supplemented media in order to select cells harbouring replicating viral RNA. Resultant G418-resistant cells were isolated and were found to be expressing affinity-tagged NS5A protein via viral replication. Viral protein complex as well as their interacting host proteins were isolated by affinity-culumn purification and analized by MudPIT in order to identify associating host factors.