A novel approach to study hepatitis C virus entry

研究丙型肝炎病毒进入的新方法

• 批准号: 6531660
• 负责人: ILA R SINGH
• 金额: $ 16.1万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6531660
• 关键词: Vesiculovirus; flow cytometry; fusion gene; gene mutation; genetic library; genetic mapping; hepatitis C virus; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): Hepatitis C virus infects 4 million Americans and an estimated 2-4% of the world's population, causing chronic hepatitis in most of those infected. A sizable fraction subsequently develops cirrhosis or hepatocellular carcinoma. This proposal describes the use of a novel approach to perform a mutational analysis of regions of the Hepatitis C virus (HCV). The objective is to delineate, at an unprecedented resolution, the role of specific sequences, both viral and cellular, in viral entry. Conventional approaches to the study of viral entry consist of morphological and biochemical studies of replication intermediates. While these approaches have been very useful for many viruses, they suffer from some drawbacks. Since viral entry is an inefficient process, with only one out of hundreds or thousands of virions following the productive pathway of infection, the non-productive particles tend to severely obscure morphological analysis and limit interpretation of biochemical studies. It also becomes difficult to get an adequate signal with physiologically relevant multiplicities of infection. This is especially pertinent to the study of the early steps of viral infection, before there is any amplification from viral expression. These problems make it necessary to complement conventional studies with the genetic approach of making and analyzing mutations that disrupt viral functions. The mutational approach is a proven and well-established strategy for the study of gene function. However, most current methods involve the isolation, storage and characterization of each mutant separately, making the process very time consuming and labor intensive. Genetic footprinting is a novel method that allows for efficient construction and parallel functional analysis of thousands of mutations in a cloned gene. The specific aims of this research are: 1) Creation of libraries of mutations in the E1 and E2 envelope glycoproteins of HCV, using a transposon-based mutagenesis method, followed by their analysis. 2) Genetic footprinting of the HCV IRES, to determine regions necessary for its function. Preliminary data showing the successful generation of libraries is presented. These libraries will be analyzed en masse to determine what regions of the glycoproteins are necessary for viral binding and fusion, and to determine what regions of the IRES are essential for translation of viral proteins. A comprehensive and detailed knowledge of the process of HCV entry gained from this study will be important for understanding the mechanism of viral infection and for the development of new preventive and therapeutic approaches against HCV.

描述（由申请人提供）：丙型肝炎病毒感染了 400 万美国人，估计占世界人口的 2-4%，导致大多数感染者患上慢性肝炎。相当大一部分随后发展为肝硬化或肝细胞癌。该提案描述了使用一种新方法对丙型肝炎病毒 (HCV) 区域进行突变分析。目标是以前所未有的分辨率描绘病毒和细胞特定序列在病毒进入中的作用。研究病毒进入的传统方法包括复制中间体的形态学和生化研究。虽然这些方法对于许多病毒非常有用，但它们也存在一些缺点。由于病毒进入是一个低效的过程，只有数百或数千个病毒颗粒中的一个遵循生产性感染途径，因此非生产性颗粒往往会严重掩盖形态分析并限制生化研究的解释。获得与生理相关的感染多重性相关的足够信号也变得困难。这与病毒表达放大之前病毒感染早期步骤的研究尤其相关。这些问题使得有必要用产生和分析破坏病毒功能的突变的遗传方法来补充传统研究。突变方法是一种经过验证且行之有效的基因功能研究策略。然而，目前的大多数方法都涉及对每个突变体分别进行分离、储存和表征，使得该过程非常耗时且费力。遗传足迹是一种新颖的方法，可以对克隆基因中的数千个突变进行有效构建和并行功能分析。本研究的具体目标是： 1) 使用基于转座子的诱变方法创建 HCV E1 和 E2 包膜糖蛋白突变文库，然后进行分析。 2) HCV IRES 的基因足迹，以确定其功能所需的区域。提供了显示文库成功生成的初步数据。将对这些文库进行整体分析，以确定糖蛋白的哪些区域对于病毒结合和融合是必需的，并确定 IRES 的哪些区域对于病毒蛋白的翻译是必需的。从这项研究中获得的关于丙型肝炎病毒进入过程的全面而详细的了解对于理解病毒感染的机制以及开发针对丙型肝炎病毒的新的预防和治疗方法非常重要。