MECHANISM OF HEPACIVIRUS REPLICASE ASSEMBLY

在美国，丙型肝炎病毒（HCV）感染了大约400万人，是急性和慢性肝病的主要原因，通常导致肝硬化和肝细胞癌。负责HCV基因组复制的病毒因子知之甚少，需要对这些因子进行详细的生化表征，以帮助合理开发抗HCV治疗方法。HCV RNA解旋酶NS3被认为对病毒复制至关重要。目前，还没有严格的RNA解旋酶活性的生化模型，这使得这类酶成为最不为人所知的病毒因子之一。本研究计划的目标是全面表征NS3的生化机制，包括其与底物和其他HCV蛋白的相互作用。利用酵母二代和三代杂交系统的遗传方法被用于鉴定丙型肝炎病毒蛋白之间以及与丙型肝炎病毒RNA特定区域之间的新的相互作用。生物化学方法被用来确定控制这种相互作用的特异性和热力学参数。RNA解绕NS3正在研究使用一种新的预稳态分析，其中酶以化学计量学的方式组装在明确的寡核苷酸底物上。通过这个实验，我们将通过测量酶的活性形式来确定NS3是否通过化学计量学或催化机制起作用。第二项新检测将用于确定NS3在单链核酸底物上的易位是否存在方向性偏倚。这些实验的结果将为开发NS3的最小动力学机制提供框架，这对于定量了解该酶的功能是必要的。我们将使用核酸足迹和蛋白质- dna交联的组合来测试几种拟议的易位和解绕机制。关键氨基酸在NS3功能中的具体作用也将通过定点诱变和x射线晶体学来解决。初步数据表明NS4b蛋白与NS4a相互作用，已知NS4a与NS3形成紧密复合物。从而确定NS4b对NS3活性的影响。该项目的结果将以定量的方式确定和表征NS3、其底物和其他HCV蛋白之间新的特异性相互作用。这项工作将为我们利用生物学相关蛋白和RNA在体外复制HCV的长期目标迈出第一步。

Hepatitis C virus (HCV) infects approximately 4 million persons in the U.S. and is a major cause of acute and chronic liver disease, often leading to cirrhosis and hepatocellular carcinoma. The viral factors that are responsible for replication of the HCV genome are poorly understood, and detailed biochemical characterization of these factors is needed to aid in rational development of anti-HCV therapies. The HCV RNA helicase, NS3, is believed to be essential for viral replication. Currently, no rigorous, biochemical models for RNA helicase activity exist, making this class of enzymes one of the least understood viral factors. The goal of this research proposal is to fully characterize the biochemical mechanism of NS3 including its interaction with substrates and other HCV proteins. Genetic methods using the yeast two and three hybrid systems are being applied to identify novel interactions among HCV proteins as well as with specific regions of HCV RNA. Biochemical methods are being used to determine the specificity and thermodynamic parameters that govern such interactions. RNA unwinding by NS3 is being studied using a new pre-steady-state assay in which the enzyme is assembled in a stoichiometric fashion on well-defined oligonucleotide substrates. Using this assay, we will determine whether NS3 functions through a stoichiometric or catalytic mechanism by measuring the active form of the enzyme. A second new assay will be applied to determine whether there exists a directional bias in translocation of NS3 on single-stranded nucleic acid substrates. The results from these experiments will provide the frame work in which to develop a minimal kinetic mechanism of NS3 which is necessary for quantitative understanding of the function of this enzyme. We will test several proposed mechanisms for translocation and unwinding using a combination of nucleic acid footprinting and protein-DNA crosslinking. The specific role of key amino acids in the function of NS3 will also be addressed using site-directed mutagenesis and x-ray crystallography. Preliminary data suggests that NS4b protein interacts with NS4a, which is known to form a tight complex with NS3. Thus, the influence of NS4b on the activity of NS3 will be determined. Results from this project will identify and characterize novel, specific interactions among NS3, its substrates, and other HCV proteins in a quantitative manner. This work will provide the initial step toward our long term goal of recapitulating HCV replication in vitro using biologically relevant proteins and RNA.