CELL CULTURE-BASED STUDIES OF HCV PATHOGENESIS

基于细胞培养的 HCV 发病机制研究

• 批准号: 6806552
• 负责人: Michael M.C. Lai
• 金额: $ 32.54万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6806552
• 关键词: B lymphocyte; DNA damage; DNA repair; DNA replication; apoptosis; cell line; flow cytometry; gene mutation; hepatitis; hepatitis C virus; immunoglobulin genes; liver cells; molecular oncology; molecular pathology; nitric oxide; oncology; polymerase chain reaction; tissue /cell culture; virus RNA; virus cytopathogenic effect

DESCRIPTION (provided by applicant): The overall objective of this project is to understand the mechanism of the pathogenesis and oncogenesis of hepatitis C virus (HCV). HCV is one of the major causes of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). It is also associated with B cell oligoclonal proliferation and non-Hodgkin's B cell lymphoma in certain geographical regions. The study of HCV has been hampered by the lack of an efficient tissue culture system for propagating HCV. Our laboratory recently established a B cell culture system for HCV infection, which allows the in vitro infection of culture cells and persistent production of HCV particles. Our preliminary findings indicate that HCV infection induces double-strand DNA breaks and apoptosis and enhances mutation frequency of cellular genes, suggesting that HCV induces a mutator phenotype. Our system thus opens up a new and unique approach for studying HCV biology in the context of complete viral infection and replication. Our specific aims for this project are: SPECIFIC AIM 1. Characterization of the biological properties of HCV-infected B cells. (a) Apoptosis associated with HCV infections, In particular, we will study the role of nitric oxide in the induction of double-strand DNA breaks, and apoptosis and its effect on HCV replication will be studied. We will also study, the mechanism of iNOS induction and the role of the active oxygen species in HCV infection. (b) The mutagenic properties of HCV infection. We will examine whether HCV infection increases the mutation frequency of cellular DNAs, causes chromosomal breaks and/or inhibits DNA repair mechanisms. (c) The co-evolution of the immunoglobulin gene and viral RNA sequences during in vitro passages. This study will contribute to the understanding of whether B cell properties are affected by HCV infection. specific aim 2. Identification of the viral gene products responsible for the various biological properties studied in Specific Aim 1. To understand the mechanism of the biological changes induced by HCV. we will examine the ability of the individual HCV gene products to induce the phenomena described in Specific Aim 1. Individual viral genes will be constructed and expressed in B cells and hepatocyte cell lines. The various parameters of apoptosis and DNA mutations will be examined. These include iNOS induction, double-strand DNA breaks, and induction of error-prone DNA polymerases. The possible involvement of extracellular E2 protein in causing these effects will also be examined.

描述（由申请人提供）：本项目的总体目标是了解丙型肝炎病毒（HCV）的发病机制和肿瘤发生机制。HCV是慢性肝炎、肝硬化和肝细胞癌（HCC）的主要病因之一。它还与某些地理区域的B细胞寡克隆增殖和非霍奇金B细胞淋巴瘤有关。由于缺乏有效的组织培养系统来繁殖HCV，阻碍了对HCV的研究。本实验室最近建立了一种用于HCV感染的B细胞培养系统，该系统允许培养细胞的体外感染和HCV颗粒的持续产生。我们的初步研究结果表明，HCV感染诱导双链DNA断裂和细胞凋亡，并提高细胞基因的突变频率，表明HCV诱导的增变表型。因此，我们的系统开辟了一个新的和独特的方法来研究HCV生物学的背景下，完整的病毒感染和复制。我们在这个项目中的具体目标是：具体目标1。HCV感染的B细胞的生物学特性的表征。(a)细胞凋亡与HCV感染相关，特别是，我们将研究一氧化氮在诱导双链DNA断裂中的作用，并研究细胞凋亡及其对HCV复制的影响。我们还将研究iNOS的诱导机制以及活性氧在HCV感染中的作用。(b)丙型肝炎病毒感染的致突变性。我们将研究HCV感染是否会增加细胞DNA的突变频率，导致染色体断裂和/或抑制DNA修复机制。(c)体外传代过程中免疫球蛋白基因和病毒RNA序列的共同进化。本研究将有助于了解HCV感染是否影响B细胞的特性。具体目标2.鉴定负责特定目的1中研究的各种生物学特性的病毒基因产物。了解丙型肝炎病毒（HCV）引起的生物学变化的机制。我们将检测单个HCV基因产物诱导特异性目标1中描述的现象的能力。将构建单个病毒基因并在B细胞和肝细胞系中表达。将检查细胞凋亡和DNA突变的各种参数。这些包括iNOS诱导、双链DNA断裂和易错DNA聚合酶的诱导。细胞外E2蛋白在引起这些影响的可能参与也将被检查。