MECHANISM OF HEPACIVIRUS REPLICASE ASSEMBLY

丙型肝炎病毒（HCV）在美国感染约400万人，并且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物化学表征，以帮助抗HCV疗法的合理开发。 HCV RNA解旋酶NS 3被认为是病毒复制所必需的。 目前，没有严格的，生物化学模型的RNA解旋酶活性存在，使这类酶的最不了解的病毒因子之一。 本研究计划的目标是充分表征NS 3的生化机制，包括其与底物和其他HCV蛋白的相互作用。 使用酵母双杂交和三杂交系统的遗传学方法正被应用于鉴定HCV蛋白之间以及与HCV RNA的特定区域之间的新型相互作用。 生物化学方法正在被用来确定的特异性和热力学参数，支配这种相互作用。 RNA解旋的NS 3正在研究使用一种新的预稳定状态的测定，其中的酶组装在一个化学计量的方式上定义明确的寡核苷酸底物。 使用该测定，我们将通过测量酶的活性形式来确定NS 3是否通过化学计量或催化机制起作用。 第二个新的检测方法将用于确定NS 3在单链核酸底物上的易位是否存在方向性偏倚。 这些实验的结果将提供一个框架，在其中开发一个最小的动力学机制的NS 3，这是必要的定量了解这种酶的功能。 我们将使用核酸足迹和蛋白质-DNA交联的组合来测试几种提出的易位和解旋机制。 关键氨基酸在NS 3功能中的特定作用也将使用定点诱变和X射线晶体学来解决。 初步数据表明，NS 4 b蛋白与NS 4a相互作用，已知NS 4a与NS 3形成紧密复合物。 因此，将确定NS 4 b对NS 3活性的影响。 该项目的结果将以定量的方式鉴定和表征NS 3，其底物和其他HCV蛋白之间的新型，特异性相互作用。 这项工作将为我们的长期目标提供第一步，即利用生物学相关蛋白质和RNA在体外重现HCV复制。

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.