CELL CULTURE-BASED STUDIES OF HCV PATHOGENESIS

基于细胞培养的 HCV 发病机制研究

• 批准号: 7118001
• 负责人: Michael M.C. Lai
• 金额: $ 31.87万
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118001
• 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）：该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)的主要原因之一。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。由于缺乏用于传播HCV的有效组织培养系统，HCV的研究受到阻碍。我们实验室最近建立了用于HCV感染的B细胞培养系统，该系统可以体外感染培养细胞并持续产生HCV颗粒。我们的初步研究结果表明，HCV感染会诱导双链DNA断裂和细胞凋亡，并增加细胞基因的突变频率，这表明HCV会诱导突变表型。因此，我们的系统为在完整病毒感染和复制的背景下研究 HCV 生物学开辟了一种新的、独特的方法。我们该项目的具体目标是： 具体目标 1. 表征 HCV 感染的 B 细胞的生物学特性。 (a)与HCV感染相关的细胞凋亡，特别是，我们将研究一氧化氮在诱导双链DNA断裂中的作用，并且将研究细胞凋亡及其对HCV复制的影响。我们还将研究 iNOS 诱导机制以及活性氧在 HCV 感染中的作用。 (b) HCV 感染的致突变特性。我们将检查 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) 体外传代期间免疫球蛋白基因和病毒RNA序列的共同进化。这项研究将有助于了解 B 细胞特性是否受到 HCV 感染的影响。具体目标 2. 鉴定负责在具体目标 1 中研究的各种生物学特性的病毒基因产物。了解 HCV 引起的生物学变化的机制。我们将检查单个 HCV 基因产物诱导具体目标 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。其中包括 iNOS 诱导、双链 DNA 断裂以及易错 DNA 聚合酶的诱导。细胞外 E2 蛋白是否可能参与引起这些影响也将得到检验。