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: 2728337
• 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/2728337
• 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.

慢性丙型肝炎病毒（HCV）感染导致肝硬化并发症的高频肝移植频率和长期病毒持久性。该提案的总体目的是研究HCV编码的非结构蛋白NS5A在病毒相互作用中的功能以及介导对I型干扰素（IFN）的抗性，这是这些患者唯一认可的治疗方法。 绝大多数患者在停止IFN治疗后不反应或复发。 迄今为止，实验证据表明，感染的严重程度和对IFN治疗的反应性是与亚型相关的HCV-NS5A序列变化。 HCV-NS5A是一种磷酸蛋白，被认为是病毒RNA复制酶的组成部分。 但是它在病毒生命周期中的精确作用尚不清楚。 最近，它已显示与多个细胞蛋白激酶相关。 这些激酶之一已被鉴定为干扰素（IFN）诱导的双链RNA激活蛋白激酶（PKR），而其他激酶的身份尚不清楚。 在本应用中提出了一个假设，即HCV-NS5A的内在特性的亚型变化与PKR与PKR相关联，而其他未识别的细胞激酶（S）则在差异上调节IFN信号转导途径，并可能导致HCV感染对IFN疗法的抗性。该提案的总体目标将通过以下特定目的来实现：1。通过使用使用大肠杆菌和杆状病毒表达系统表达的重组NS5A蛋白与NS5A相关的细胞激酶。 我们将通过相互作用亲和色谱方法纯化激酶，并使用许多肽作为以前针对众所周知的蛋白激酶表征的底物。 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。 将分离出在诱导型启动子下有条件表达NS5A的肝细胞癌（HEPG2）细胞系。 NS5A相关的激酶活性将与IFN治疗细胞的作用定量相关。 我们将研究NS5A的表达是否使用免疫沉淀分析和电泳迁移率转移分析干扰IFN信号通路。 3。完成特定目标1和2后，我们将确定相关激酶在体外和细胞内使用的NS5A的磷酸化位点，这是通过IFN通过二维磷酸肽映射技术和微层次分析的IFN处理的结果。