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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 -背景和目标 丙型肝炎病毒(丙型肝炎病毒)是一种终生持续感染，可引起慢性炎症性疾病，最终导致肝脏疾病的严重并发症，包括纤维化、肝硬变和肝细胞癌。作为黄病毒科肝病毒属的唯一成员，这种正链极性的单链RNA病毒在感染细胞的胞浆室中复制，并导致一种被认为是从粗面内质网延伸而来的改变的膜性结构，并与病毒RNA的活跃复制有关。尽管对这种病毒如何在细胞内建立其微环境知之甚少，但在需要产生病毒粒子作为包膜病毒的背景下，揭示丙型肝炎病毒如何处理脂代谢及其运输正成为人们非常感兴趣的问题。 -方法 将亚基因组复制子RNA导入人肝癌细胞株Huh-7，在非结构蛋白5A(NS5A)的C末端引入亲和标记多肽序列。用添加G418的培养液培养细胞，以筛选出含有复制病毒RNA的细胞。获得的G418抗性细胞被分离，并被发现通过病毒复制表达亲和标记的NS5A蛋白。用亲和层析法分离病毒蛋白复合体及其相互作用的宿主蛋白，并用MudPIT进行分析，以确定相关宿主因子。

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.