Characterization of the Hepatitis C NS5a Kinase Complex

丙型肝炎病毒感染以及与之相关的慢性肝炎、肝硬变和肝细胞癌是美国肝移植的主要原因。美国人口中约有400万丙型肝炎病毒携带者。据估计，全世界感染丙型肝炎病毒的总人数为1.7亿人，占总人口的近3%。在丙型肝炎病毒在感染者体内复制的过程中，病毒复制酶复合体的关键组成部分NS5A蛋白被一种未知的细胞激酶磷酸化。这种磷酸化也被证明发生在携带丙型肝炎病毒复制子的细胞系中，在那里已经观察到复制效率和磷酸化之间的联系。这项研究的目的是通过使用常规的层析分离和2D凝胶电泳/MALDI-TOF质谱仪来鉴定和表征导致丙型肝炎病毒NS5A蛋白磷酸化的一个或多个细胞激酶。观察到的病毒RNA复制的磷酸化的影响将使用一系列复制子突变体来检验，这些突变体表现出不同水平的NS5A磷酸化和复制能力。与NS5A相关激酶和丙型肝炎病毒复制酶复合体成分相互作用所需的NS5A蛋白区域将使用一组NS5A缺失和突变来鉴定。此外，还将检查与NS5A蛋白和NS5A激酶相关的复制复合体的成分的鉴定，并可能提供一些对丙型肝炎病毒RNA复制的基本未知要求的洞察。

Hepatitis C virus (HCV) infection and the associated chronic hepatitis, cirrhosis and hepatocellular carcinoma are the leading cause of liver transplantation in the United States. There are approximately 4 million HCV carriers in the U.S. population. Estimates place the total number of HCV infected individuals at 170 million worldwide, representing nearly 3% of the population. During the course of HCV replication in infected individuals, the NS5a protein, a key component of the viral replicase complex, is phosphorylated by an unknown cellular kinase. This phosphorylation has been shown to also occur in cell lines carrying HCV replicons, where a link between replication efficiency and phosphorylation has been observed. The studies in this proposal are designed to identity and characterize the cellular kinase or kinases responsible for the phosphorylation of the NS5a protein of HCV by the use of conventional chromatographic separations and 2D gel electrophoresis/MALDI-TOF mass spectrometry. The effects of the observed phosphorylation of viral RNA replication will be examined using a series of replicon mutants that exhibit differing levels of NS5a phosphorylation and replication competence. The region of the NS5a protein required for the interaction with the NS5a associated kinase and the HCV replicase complex components will be identified using a battery of NS5a deletions and mutants. In addition, identification of the components of the replication complex associated with the NS5a protein and NS5a kinase will be examined and may provide some insight into the largely uncharacterized requirements for HCV viral RNA replication.