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.

在美国，慢性丙型肝炎病毒(丙型肝炎病毒)感染导致肝脏移植的频率很高，这是肝硬变和病毒长期存在的并发症的结果。这项建议的总体目标是研究丙型肝炎病毒编码的非结构蛋白NS5A在病毒-宿主相互作用和介导对I型干扰素(干扰素)的耐药性中的功能，I型干扰素是这些患者唯一被批准的治疗方法。绝大多数患者在停止干扰素治疗后没有反应或复发。到目前为止的实验证据表明，感染的严重性和对干扰素治疗的反应性是与亚型相关的丙型肝炎病毒NS5A序列变异。丙型肝炎病毒NS5A是一种磷酸蛋白，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，它被证明与不止一种细胞蛋白激酶有关。其中一种已被鉴定为干扰素诱导的双链核糖核酸激活的蛋白激酶，另一种激酶(S)的身份尚不清楚。在这一应用中，提出了一种假设，即与PKR和其他未知细胞激酶(S)相关的丙型肝炎病毒NS5A固有特性的亚型变异对干扰素信号转导途径进行了不同的调节，并可能导致丙型肝炎病毒感染对干扰素治疗的耐药性。这项建议的总体目标将通过以下具体目标来实现：1.利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(S)。我们将通过相互作用亲和层析的方法来纯化该激酶(S)，并使用一些以前已知的蛋白激酶的多肽作为底物。我们将对纯化的激酶进行蛋白质微序列分析。2.剖析NS5A与未知的细胞激酶、PKR和STAT1的分子相互作用。将分离出在可诱导启动子下有条件表达NS5A的肝细胞癌(HepG2)细胞系。NS5A相关激酶活性将与干扰素治疗细胞的效果定量相关。我们将使用免疫沉淀分析和电泳迁移率改变分析来研究NS5A的表达是否干扰了干扰素信号通路。3.在完成特异性目标1和2之后，我们将通过二维磷酸肽图技术和微序列分析来确定NS5A在体外和细胞内被干扰素处理后所利用的结合蛋白的磷酸化位点。