Molecular Modulators of HCV Replication

(修订摘要)描述：调节丙型肝炎病毒(丙型肝炎病毒)复制的分子事件在很大程度上是未知的。丙型肝炎病毒非结构蛋白NS5B通过(-)链中间体合成病毒正链RNA基因组，被认为是病毒复制的关键酶，但其活性如何控制仍有许多悬而未决的问题。此外，很少有宿主细胞蛋白(S)/因子(S)和其他非结构性(NS)丙型肝炎病毒蛋白与NS5B相互作用并调节其功能、合成或周转。因此，这里提出了两个大的方面，结构-活性关系(SAR)优化研究，以设计和合成新的NS5B特异性抑制剂，以及鉴定促进NS5B相互作用的活性的细胞/病毒蛋白(S)。在目标I中，我们将探索苯丙氨酸衍生的非核苷化合物在抑制NS5B酶活性方面的潜力。这些研究将以NS5B的晶体结构及其与其他聚合酶结构的相似性为指导。NS5B含有一个疏水的C末端尾巴，有助于蛋白质的膜整合，形成与细胞骨架元件相关的复制体复合体。然而，NS5B与该复合体的细胞和其他病毒成分之间的物理和功能相互作用尚不清楚。在AIM II中，我们建议使用密码基因蛋白质组学技术来鉴定与NS5B相互作用的丙型肝炎病毒非结构蛋白。为此，我们将使用UHCV-11细胞系，诱导表达除NS5B外的整个丙型肝炎病毒开放阅读框架。控制这些相互作用的动力学参数将通过表面等离子激元共振(Biacore)得到验证。在目标III中，我们建议从正常和丙型肝炎病毒感染的肝组织中鉴定宿主细胞蛋白(S)，与NS5B相互作用，从而调节其催化功能。从NS5B的结构-功能关系及其与其他蛋白质的组合相互作用的角度对NS5B进行批判性的理解，将有助于深入了解介导丙型肝炎病毒复制的分子效应器。这将使我们能够在分子水平上了解丙型肝炎病毒的复制过程，并将进一步促进有效的抗丙型肝炎药物/抑制剂的开发。

(Revised Abstract) DESCRIPTION: The molecular events that mediate replication of hepatitis C virus (HCV) are largely obscure. The HCV nonstructural protein, NS5B, that synthesizes the viral positive strand RNA genome via (-) strand intermediate, is believed to be the key enzyme responsible for its replication but there are many unanswered questions about how its activity is controlled. Moreover, very few host cell protein(s)/factor(s) and other non-structural (NS) HCV proteins, which interact with NS5B and modulate its function, synthesis or turnover, have been identified and characterized. Proposed here, therefore, are two broad aspects, structure-activity relationship (SAR) optimization studies to design, and synthesize novel NS5B specific inhibitors and identification of cellular/viral protein(s) facilitating NS5B's activity upon interaction. In Aim I, we will explore the potential of phenylalanine-derived non-nucleoside compounds in inhibiting NS5B's enzymatic activity. These studies will be guided by the resolved crystal structures of NS5B and its similarities with other polymerase's structures. NS5B contains a hydrophobic C-terminal tail that aids in membrane integration of the protein to form a replisome complex which is associated with cytoskeletal elements. However, the physical and functional interactions between NS5B and the cellular and other viral components of this complex are poorly understood. In Aim II we propose to identify HCV nonstructural proteins interacting with NS5B using the ciphergen proteomic technology. For this, we will employ the UHCV-11 cell line, inducibly expressing the entire HCV open reading frame except NS5B. The kinetic parameters governing these interactions will be validated via surface plasmon resonance (Biacore). In Aim III, we propose to identify host cell protein(s) from normal and HCV-infected liver explants, interacting with NS5B and thereby modulating its catalytic function. A critical understanding of NS5B, in perspective of its structure-function relationships and its combinatorial interaction with other proteins, will provide insights into the molecular effectors mediating HCV replication. This will enable us to understand, at the molecular level, the replication process of hepatitis C virus and will further facilitate the development of effective drugs/inhibitors against HCV.