A novel approach to study hepatitis C virus entry

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%，大多数感染者导致慢性肝炎。相当一部分随后发展为肝硬化或肝细胞癌。本提案描述了使用一种新的方法来执行丙型肝炎病毒（HCV）区域的突变分析。目的是描述，在一个前所未有的分辨率，特定序列的作用，病毒和细胞，在病毒进入。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。虽然这些方法对许多病毒非常有用，但它们也有一些缺点。由于病毒进入是一个效率低下的过程，数百或数千个病毒粒子中只有一个遵循感染的生产途径，非生产颗粒往往严重模糊形态分析并限制生化研究的解释。在感染的生理相关的多重性中也很难获得足够的信号。这尤其适用于研究病毒感染的早期步骤，在病毒表达有任何扩增之前。这些问题使得有必要用制造和分析破坏病毒功能的突变的遗传方法来补充传统的研究。突变方法是研究基因功能的一种行之有效的策略。然而，目前的大多数方法涉及分离、储存和表征每个突变体，使这一过程非常耗时和劳动密集型。遗传足迹是一种新颖的方法，它允许对克隆基因中的数千个突变进行有效的构建和并行功能分析。本研究的具体目的是：1)利用基于转座子的诱变方法建立HCV E1和E2包膜糖蛋白突变文库，然后对其进行分析。2) HCV IRES的遗传足迹，以确定其功能所需的区域。给出了显示库生成成功的初步数据。这些文库将被大量分析，以确定糖蛋白的哪些区域是病毒结合和融合所必需的，以及IRES的哪些区域是病毒蛋白翻译所必需的。从这项研究中获得的关于HCV进入过程的全面和详细的知识将对理解病毒感染的机制和开发新的HCV预防和治疗方法具有重要意义。

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.