CELL CULTURE-BASED STUDIES OF HCV PATHOGENESIS

描述(申请人提供)：本项目的总体目标是了解丙型肝炎病毒(丙型肝炎病毒)的发病机制和致癌机制。丙型肝炎病毒是慢性肝炎、肝硬变和肝细胞癌的主要病因之一。它还与某些地理区域的B细胞寡克隆性增殖和非霍奇金B细胞淋巴瘤有关。由于缺乏有效的组织培养系统来繁殖丙型肝炎病毒，对丙型肝炎病毒的研究一直受到阻碍。我们实验室最近建立了一套用于丙型肝炎病毒感染的B细胞培养系统，该系统允许体外培养细胞感染并持续产生丙型肝炎病毒颗粒。我们的初步发现表明，丙型肝炎病毒感染可诱导双链DNA断裂和细胞凋亡，并提高细胞基因的突变频率，提示丙型肝炎病毒可诱导突变表型。因此，我们的系统为在完全病毒感染和复制的背景下研究丙型肝炎病毒生物学开辟了一种新的和独特的方法。我们这个项目的具体目标是：特定目的1.丙型肝炎病毒感染的B细胞的生物学特性的表征。(A)与丙型肝炎病毒感染相关的细胞凋亡，特别是一氧化氮在诱导双链DNA断裂中的作用，以及细胞凋亡及其对丙型肝炎病毒复制的影响。我们还将研究诱导型一氧化氮合酶的诱导机制以及活性氧在丙型肝炎病毒感染中的作用。(B)丙型肝炎病毒感染的致突变特性。我们将研究丙型肝炎病毒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)免疫球蛋白基因和病毒RNA序列在体外传代过程中的共同进化。这项研究将有助于了解丙型肝炎病毒感染是否影响B细胞的特性。特定目的2.鉴定与特定目的研究中所研究的各种生物学特性有关的病毒基因产物1.了解丙型肝炎病毒引起生物变化的机制。我们将检测单个丙型肝炎病毒基因产物诱导特定目的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。将检测细胞凋亡和DNA突变的各种参数。这些包括诱导型一氧化氮合酶的诱导，双链DNA断裂，以及容易出错的DNA聚合酶的诱导。细胞外的E2蛋白可能参与引起这些效应也将被研究。

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.