VIRUS/HOST INTERACTIONS MODULATED BY HEPATITIC C VIRUS

丙型肝炎病毒调节的病毒/宿主相互作用

• 批准号: 6171117
• 负责人: Radhakrishnan Padmanabhan
• 金额: $ 7.5万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171117
• 关键词: affinity chromatography; biological signal transduction; cell line; enzyme mechanism; gel mobility shift assay; hepatitis C virus; host organism interaction; immunoprecipitation; interferons; intermolecular interaction; phosphorylation; protein kinase; protein purification; recombinant proteins; transfection; virus infection mechanism; virus protein

Chronic hepatitis C virus (HCV) infections leads to a high frequency of liver transplants in the U.S. as a result of complications of liver cirrhosis and long term virus persistence. The overall objective of this proposal is to investigate the function of the HCV-encoded nonstructural protein NS5A in virus- host interactions and in mediating resistance to type I interferon (IFN), which is the only approved therapy for these patients. Large majority of patients do not respond to or relapse after cessation of IFN therapy. Experimental evidence to date suggest that severity of infection and responsiveness to IFN therapy are subtype-related HCV-NS5A sequence variations. HCV- NS5A is a phosphoprotein and is thought to be a component of viral RNA replicase. But its precise role in the virus life cycle is unknown. Recently, it has been shown to associate with more than one cellular protein kinases. One of these kinases has been identified to be interferon (IFN)-inducible double-stranded RNA activated protein kinase (PKR) and the identity of other kinase(s) is unknown. A hypothesis is proposed in this application that the subtype variations in the intrinsic properties of HCV-NS5A to associate with PKR and other unidentified cellular kinase(s) modulate differentially the IFN signal transduction pathways and may contribute to resistance of HCV infections to IFN therapy. The overall objective of this proposal will be accomplished through the following Specific Aims: 1. To identify the cellular kinase(s) that associates with NS5A by using recombinant NS5A proteins expressed using E. coli and baculovirus expression systems. We will purify the kinase(s) by interaction affinity chromatographic methods and use a number of peptides as substrates which are previously characterized for well known protein kinases. We will carry out protein microsequence analysis of the purified kinase. 2. To dissect the molecular interactions of NS5A with the unidentified cellular kinase, PKR, and STAT1. A hepatocellular carcinoma (HepG2) cell line conditionally expressing NS5A under an inducible promoter will be isolated. The NS5A-associated kinase activities will be quantitatively correlated with the effect of IFN treatment of cells. We will investigate whether expression of NS5A interferes with IFN signaling pathways using immunoprecipitation analyses and electrophoretic mobility shift assays. 3. After completion of specific aims 1 and 2, we will identify the phosphorylation sites of NS5A utilized by the associated kinase in vitro as well as intracellularly as a result of IFN treatment by two- dimensional phosphopeptide mapping techniques and microsequence analyses.

由于肝硬化并发症和长期病毒持续存在，慢性丙型肝炎病毒（HCV）感染导致美国肝移植频率较高。该提案的总体目标是研究 HCV 编码的非结构蛋白 NS5A 在病毒-宿主相互作用以及介导 I 型干扰素 (IFN) 耐药性中的功能，而 I 型干扰素 (IFN) 是这些患者唯一获批的治疗方法。 大多数患者对干扰素治疗没有反应或在停止干扰素治疗后复发。 迄今为止的实验证据表明，感染的严重程度和对 IFN 治疗的反应是与亚型相关的 HCV-NS5A 序列变异。 HCV-NS5A 是一种磷蛋白，被认为是病毒 RNA 复制酶的组成部分。 但它在病毒生命周期中的确切作用尚不清楚。 最近，它已被证明与不止一种细胞蛋白激酶相关。 其中一种激酶已被鉴定为干扰素 (IFN) 诱导型双链 RNA 激活蛋白激酶 (PKR)，其他激酶的身份尚不清楚。 在本申请中提出了一个假设，即HCV-NS5A的内在特性的亚型变异与PKR和其他未鉴定的细胞激酶相关，差异性地调节IFN信号转导途径，并且可能有助于HCV感染对IFN治疗的抵抗。该提案的总体目标将通过以下具体目标来实现： 1. 通过使用大肠杆菌和杆状病毒表达系统表达的重组 NS5A 蛋白来鉴定与 NS5A 相关的细胞激酶。 我们将通过相互作用亲和层析方法纯化激酶，并使用许多肽作为底物，这些肽先前已针对众所周知的蛋白激酶进行了表征。 我们将对纯化的激酶进行蛋白质微序列分析。 2. 剖析 NS5A 与未鉴定的细胞激酶 PKR 和 STAT1 的分子相互作用。 将分离在诱导型启动子下条件性表达 NS5A 的肝细胞癌 (HepG2) 细胞系。 NS5A相关激酶活性将与细胞的IFN处理效果定量相关。 我们将使用免疫沉淀分析和电泳迁移率变动分析来研究 NS5A 的表达是否干扰 IFN 信号通路。 3. 完成具体目标 1 和 2 后，我们将通过二维磷酸肽图谱技术和微序列分析，确定 IFN 治疗导致的体外和细胞内相关激酶利用的 NS5A 磷酸化位点。