IDENTIFICATION OF NOVEL HOST FACTORS INTERACTING WITH NS5A PROTEIN OF HCV

与 HCV NS5A 蛋白相互作用的新型宿主因子的鉴定

• 批准号: 8171374
• 负责人: ALEEM SIDDIQUI
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171374
• 关键词: Affinity; Cell Line; Cells; Chronic; Cirrhosis; Complication; Computer Retrieval of Information on Scientific Projects Database; Disease; Family; Fibrosis; Flaviviridae; Funding; Grant; Hepatitis C virus; Hepatitis C-Like Viruses; Human; Infection; Inflammatory; Institution; Integration Host Factors; Internet; Liver diseases; Methods; Nonstructural Protein; Patients; Primary carcinoma of the liver cells; Production; Proteins; Replicon; Research; Research Personnel; Resistance; Resources; Rough endoplasmic reticulum; Source; Structure; United States National Institutes of Health; Viral; Viral Proteins; Virion; Virus; antibiotic G 418; hepatoma cell; interest; lipid metabolism; member; novel; protein aminoacid sequence; protein complex; trafficking; viral RNA; virus envelope

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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 - 背景和目标 已知丙型肝炎病毒（HCV）建立终身持续感染，导致患者慢性炎症性疾病，最终导致肝病的严重并发症，包括纤维化，肝硬化和肝细胞癌。 作为Flaviviridae家族的肝病属的唯一成员，这种ssRNA病毒在受感染细胞的胞质室中复制，并导致一种称为“膜状网络”的变化的膜结构变化，被认为可以从粗糙的腹膜重复和重复进行重复的腹膜上延伸。尽管对这种病毒如何建立其在细胞内部的微观病毒的知识知之甚少，但揭示HCV如何处理脂质代谢及其在对病毒生产作为信封病毒的需求的背景下，这是引起人们极大的兴趣。 -方法 人肝癌细胞系HuH-7被亚基复制子RNA转染，在该复制子RNA中，在非结构蛋白5a（NS5A）的C端区域中引入了亲和力标记肽序列。将转染的细胞用G418补充培养基培养，以选择具有复制病毒RNA的细胞。分离出结果的G418耐药细胞，并发现通过病毒复制表达具有亲和力标记的NS5A蛋白。病毒蛋白复合物及其相互作用的宿主蛋白通过亲和力 - 脉络纯化分离，并通过Mudpit进行分析，以鉴定关联的宿主因子。