Novel antviral targets in Hepatitis C virus NS4B protein

描述（由申请人提供）：该提案概述了一个5年的培训计划，以促进博士Einav的职业发展作为一个独立的学术医生，科学家。Einav博士最近在斯坦福大学完成了传染病的临床培训。拟议的项目利用斯坦福大学现有的优秀技术和智力资源进行丙型肝炎病毒（HCV）的研究。Glenn博士和Quake博士将担任该项目的共同导师，他们分别是国际公认的HCV分子病毒学和微流体学权威。目前的治疗方法对全世界1.7亿HCV感染者中的大多数人来说是不够的。我们的长期目标是更好地了解HCV的分子病毒学，并将这些知识转化为新的抗病毒策略。该项目的目标是更好地了解非结构4 B（NS 4 B）在HCV RNA复制和细胞转化中的作用，并设计新的抗HCV策略。我们的总体假设是，NS 4 B的两个不同的迄今未被认识到的功能，核苷酸结合和水解和HCV RNA结合，是每一个必不可少的介导NS 4 B在HCV复制中的作用，前者也是必不可少的介导NS 4 B在细胞转化中的作用。因此，旨在破坏这些功能的方法可能被用于抑制HCV复制。核苷酸结合基序（NBM）的药理学抑制也可以抑制相关的肝癌。我们的初步结果表明，NS 4 B结合和水解GTP和结合HCV RNA。遗传上破坏保守的NBM和RNA结合结构域会损害其生化活性并显著抑制HCV复制。此外，NS 4 B可以独立于Ha-ras基因介导小鼠的细胞转化和肿瘤形成。具体目的和研究设计：1）通过定义介导这些功能的额外关键元件和通过确定RNA结合的底物特异性来表征NS 4 B及其NBM的RNA结合。RNA结合的生化分析将使用新的微流体平台进行。2)通过确定破坏NBM的各种元件和RNA结合元件的遗传效应来确定NS 4 B在HCV RNA复制中的作用。3)通过确定NBM在介导该活性中的作用，通过使用序列分析确定NS 4 B在引起人类肝癌中的作用，以及通过表征NS 4 B转化克隆中的基因表达谱，来确定NS 4 B在恶性转化中的作用。总的来说，迫切需要更有效的治疗方法来对抗HCV，这是病毒性肝炎的主要原因。我们希望这项研究将有助于将我们的分子病毒学研究结果转化为新型抗HCV药物的开发。后者不仅可以补充目前的药物鸡尾酒，而且通过攻击新的靶点，有望提高所产生的组合的治疗效果。

DESCRIPTION (provided by applicant): The proposal outlines a 5 year training plan to facilitate Dr. Einav's career development as an independent academic physician-scientist. Dr. Einav recently completed clinical training in infectious diseases at Stanford University. The proposed project takes advantage of outstanding technical and intellectual resources available at Stanford for the study of hepatitis C virus (HCV). Drs. Glenn and Quake who will serve as co-mentors for the project are both internationally recognized authorities on the molecular virology of HCV, and microfluidics, respectively. Current therapies are inadequate for most of the 170 million people infected with HCV worldwide. Our long-term objectives are to better understand the molecular virology of HCV and translate this knowledge into new antiviral strategies. This project's goal is to better understand non-structural 4B's (NS4B) role in HCV RNA replication and cellular transformation and enable the design of new anti-HCV strategies. Our overall hypothesis is that two different heretofore unrecognized functions of NS4B; nucleotide binding and hydrolysis and HCV RNA binding, are each essential for mediating NS4B's role in HCV replication, and the former is also essential for mediating NS4B's role in cellular transformation. Thus approaches designed to disrupt these functions may be potentially used to inhibit HCV replication. Pharmacologic inhibition of the nucleotide binding motif (NBM) may also inhibit the associated hepatoma. Our preliminary results suggest that NS4B binds and hydrolyzes GTP and binds HCV RNA. Genetically disrupting the conserved NBM and RNA binding domain impairs their biochemical activity and dramatically inhibits HCV replication. Furthermore, NS4B can mediate cellular transformation and tumor formation in mice independently of the Ha-ras gene. Specific aims and research design: 1) To characterize RNA binding by NS4B and its NBM by defining additional critical elements that mediate these functions and by determining the substrate specificity of the RNA binding. Biochemical analysis of RNA binding will be done using a novel microfluidics platform. 2) To define NS4B's role in HCV RNA replication by determining the genetic effects of disruption of the various elements of the NBM and RNA binding elements. 3) To define NS4B's role in malignant transformation by determining the role of the NBM in mediating this activity, by determining the role of NS4B in causing hepatoma in humans using sequence analysis, and by characterizing gene expression profiling in NS4B transformed clones. Taken together, more effective therapies are urgently needed against HCV, a major cause of viral hepatitis. We hope that this study will help translate our molecular virology findings into the development of new classes of anti-HCV agents. The latter could not only complement current drug cocktails, but by attacking novel targets would hopefully increase the therapeutic efficacy of the resulting combination.