VIRUS/HOST INTERACTIONS MODULATED BY HEPATITIC C VIRUS

在美国，慢性丙型肝炎病毒（HCV）感染导致肝移植的频率很高，这是肝硬化并发症和病毒长期存在的结果。该提案的总体目标是研究hcv编码的非结构蛋白NS5A在病毒-宿主相互作用和介导对I型干扰素（IFN）的耐药性中的功能，IFN是唯一被批准用于这些患者的治疗方法。绝大多数患者在停止干扰素治疗后无反应或复发。迄今为止的实验证据表明，感染的严重程度和对IFN治疗的反应性是亚型相关的HCV-NS5A序列变异。HCV- NS5A是一种磷酸化蛋白，被认为是病毒RNA复制酶的一个组成部分。但它在病毒生命周期中的确切作用尚不清楚。最近，它已被证明与一种以上的细胞蛋白激酶相关。其中一种激酶已被鉴定为干扰素（IFN）诱导的双链RNA激活蛋白激酶（PKR），其他激酶的身份尚不清楚。本研究提出了一种假设，即HCV- ns5a与PKR和其他未识别的细胞激酶相关的内在特性的亚型变异对IFN信号转导通路的调节存在差异，并可能导致HCV感染对IFN治疗产生耐药性。本提案的总体目标将通过以下具体目标来实现：利用大肠杆菌和杆状病毒表达系统表达的重组NS5A蛋白，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，这些底物以前被描述为已知的蛋白激酶。我们将对纯化的激酶进行蛋白微序列分析。2. 分析NS5A与未知细胞激酶PKR和STAT1的分子相互作用。在诱导启动子下，分离出一株条件表达NS5A的HepG2细胞系。ns5a相关激酶的活性与IFN对细胞的作用有一定的定量关系。我们将使用免疫沉淀分析和电泳迁移转移试验来研究NS5A的表达是否干扰IFN信号通路。3. 在完成特定目标1和2之后，我们将通过二维磷酸肽定位技术和微序列分析，在体外和细胞内鉴定IFN处理后相关激酶利用的NS5A磷酸化位点。

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.