Molecular Modulators of HCV Replication

HCV 复制的分子调节剂

• 批准号: 6740753
• 负责人: Neerja Kaushik-Basu
• 金额: $ 26.5万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740753
• 关键词: antiviral agents; drug design /synthesis /production; hepatitis C virus; host organism interaction; microarray technology; protein protein interaction; protein structure function; site directed mutagenesis; surface plasmon resonance; tissue /cell culture; virus protein; virus replication

(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.

（修订摘要）描述：介导乙型肝炎病毒复制（HCV）的分子事件在很大程度上是晦涩的。 HCV非结构蛋白NS5B，通过（ - ）链中间体合成病毒阳性链RNA基因组，被认为是负责复制的关键酶，但有关其活性如何控制的未解决的问题。此外，已经确定和表征了与NS5B相互作用并调节其功能，合成或周转的其他非结构性（NS）HCV蛋白的宿主细胞蛋白（S）/因子（S）和其他非结构性（NS）HCV蛋白。因此，这里提出的是两个广泛的方面，结构活性关系（SAR）优化研究研究并合成了新型NS5B特异性抑制剂，并鉴定了相互作用时NS5B活性的细胞/病毒蛋白（S）。在目标I中，我们将探讨苯丙氨酸来源的非核苷化合物在抑制NS5B的酶活性中的潜力。这些研究将以NS5B的分辨晶体结构及其与其他聚合酶结构的相似性为指导。 NS5B包含一个疏水C末端尾巴，有助于蛋白质的膜整合，形成与细胞骨架元素相关的重卵体复合物。但是，对该复合物的NS5B与细胞和其他病毒成分之间的物理和功能相互作用知之甚少。在AIM II中，我们建议使用CIPHERGEN蛋白质组学技术鉴定与NS5B相互作用的HCV非结构蛋白。为此，我们将采用UHCV-11细胞系，诱导表达除NS5B以外的整个HCV开放式阅读框架。控制这些相互作用的动力学参数将通过表面等离子体共振（BIACORE）验证。在AIM III中，我们建议从正常和HCV感染的肝外植体中鉴定宿主细胞蛋白，从而与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联合相互作用的角度，对NS5B的批判性理解将提供对介导HCV复制的分子效应子的见解。这将使我们能够在分子水平上理解丙型肝炎病毒的复制过程，并将进一步促进针对HCV的有效药物/抑制剂的开发。