Characterization of the Hepatitis C NS5a Kinase Complex

在美国，丙型肝炎病毒（HCV）感染以及相关的慢性肝炎、肝硬化和肝细胞癌是肝移植的主要原因。美国人口中大约有400万HCV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，占人口的近3%。在感染个体的HCV复制过程中，病毒复制酶复合体的关键成分NS5a蛋白被一种未知的细胞激酶磷酸化。这种磷酸化也发生在携带HCV复制子的细胞系中，在那里观察到复制效率和磷酸化之间的联系。本研究旨在通过常规色谱分离和2D凝胶电泳/MALDI-TOF质谱法鉴定和表征HCV NS5a蛋白磷酸化的细胞激酶或激酶。我们将使用一系列表现出不同水平的NS5a磷酸化和复制能力的复制子突变体来研究所观察到的病毒RNA复制磷酸化的影响。NS5a蛋白与NS5a相关激酶和HCV复制酶复合物组分相互作用所需的区域将通过一系列NS5a缺失和突变体进行鉴定。此外，鉴定与NS5a蛋白和NS5a激酶相关的复制复合体的成分将被检查，并可能为HCV病毒RNA复制的大部分未表征的需求提供一些见解。

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.