Hepatitis C virus and liver fibrogenesis

描述(申请人提供)：本提案的主要目的是了解丙型肝炎病毒感染所致肝纤维化的分子机制(S)。丙型肝炎病毒是世界范围内慢性肝病的主要原因。丙型肝炎病毒感染通常会导致高达60%-80%的感染者的慢性肝炎，并进展为肝纤维化、肝硬变和肝细胞癌。慢性丙型肝炎肝损伤和肝纤维化的机制尚不清楚。丙型肝炎病毒基因组是一种正向单链RNA分子，编码约3000个氨基酸的多蛋白前体，被宿主和病毒编码的蛋白酶翻译后切割成结构蛋白(核心、E1和E2)和非结构蛋白(NS2、NS3、NS4A、NS4B、NS5A、NS5B)。最近，我们发现在表达丙型肝炎病毒非结构蛋白的培养细胞中ROS水平升高。这一建议的主要假设是，丙型肝炎病毒诱导的氧化应激和钙信号诱导转化生长因子-21(TGF-21)的合成和激活，及其在丙型肝炎病毒复制和肝纤维化中的潜在作用。为了验证这一假设，目标是利用最近描述的丙型肝炎病毒细胞培养感染系统来描述诱导转化生长因子-21的信号通路。其具体目的是：1)明确不同的丙型肝炎病毒(1b型和2a型)激活转化生长因子-21的动力学。2)分析丙型肝炎病毒诱导的钙信号和氧化应激在刺激转化生长因子-21合成中的作用。3)明确细胞激酶和转录因子在反式调节转化生长因子-21基因表达中的作用。4)研究丙型肝炎病毒诱导的内源性生物活性转化生长因子-21对丙型肝炎病毒复制的影响。通过分析丙型肝炎病毒感染细胞以及抗氧化剂和钙离子螯合剂处理的细胞中转化生长因子-21的RNA和蛋白水平，将监测丙型肝炎病毒诱导的氧化应激和钙信号转导对转化生长因子-21表达的刺激。接下来，将通过使用定量RT-PCR测量丙型肝炎病毒RNA水平来监测转化生长因子-21对丙型肝炎病毒复制的调节。采用两步亲和纯化的方法，从用转化生长因子-21 siRNA沉默的表达细胞中纯化丙型肝炎病毒核糖核蛋白复合体(RNP)，以检测其对转化生长因子-21的反应。建议的研究将对导致肝纤维化、肝硬变的促纤维化因子的激活机制产生新的见解，并为描绘肝癌前的途径铺平道路。公共卫生相关性：丙型肝炎病毒感染通常导致慢性肝炎，进而发展为肝纤维化、肝硬变和肝细胞癌。丙型肝炎病毒感染的肝细胞表现出氧化应激，这可能在丙型肝炎病毒感染和转化生长因子-21激活之间提供了联系，从而导致纤维化的进展。这些研究产生的信息有可能为丙型肝炎病毒诱导的氧化应激和钙信号如何诱导促纤维化因子、转化生长因子-21、肝纤维化和最终肝癌提供线索。

DESCRIPTION (provided by applicant): The major goal of this proposal is to understand the molecular mechanism(s) of liver fibrosis by hepatitis C virus (HCV) infection. Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV infection often leads to chronic hepatitis in up to 60-80% of infected adults and progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. The mechanisms underlying the liver injury and fibrosis in chronic hepatitis C are unclear. The HCV genome is a positive-sense single stranded RNA molecule that encodes a polyprotein precursor of ~3000 amino acids which is post- translationally cleaved by host and virus-encoded proteases into structural proteins (core, E1, and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). Recently, we have shown the elevated levels of ROS in cultured cells expressing HCV nonstructural proteins. This proposal's main hypothesis is that HCV-induced oxidative stress and Ca2+ signaling induce the synthesis and activation of transforming growth factor-21 (TGF-21) and its potential role in HCV replication and liver fibrosis. To test this hypothesis, the goal is to delineate the signaling pathways in induction of TGF-21 using recently described HCV cell culture infection system. The specific aims are to: 1) To define the kinetics of TGF-21 activation by different HCV genotypes (type 1b vs. 2a). 2) To analyze the role of HCV-induced Ca2+ signaling and oxidative stress in stimulating TGF-21 synthesis. 3) To define the role of cellular kinases and transcription factors on trans-regulation of TGF-21 gene expression. 4) To determine the effect of HCV-induced endogenous bioactive TGF-21 on HCV replication. The stimulation of TGF- 21 expression by HCV-induced oxidative stress and Ca2+ signaling, will be monitored by analyzing the RNA and protein levels of TGF- 21 from HCV infected cells and those treated with antioxidant and Ca2+ chelators. Next, TGF- 21 mediated regulation of HCV replication will be monitored by measuring the HCV RNA levels using quantitative RT- PCR. The assembly of HCV ribonucleoprotein complex (RNP) in response to TGF-21 will be examined by using two-step affinity purification of RNP complexes from HCV expressing cells silenced with TGF-21 siRNA. The proposed studies will yield novel insights into mechanisms of activation of profibrogenic factors that leads to hepatic fibrosis, cirrhosis and pave the way for delineating the pathways preceding liver cancer. PUBLIC HEALTH RELEVANCE: HCV infection often leads to chronic hepatitis which progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. HCV infected hepatocytes display oxidative stress which may provide a link between HCV infection and activation of TGF-21 that leads to progression of fibrosis. The information resulting from these investigations has a potential to provide clues to how HCV-induced oxidative stress and calcium signaling may induce profibrogenic factor, TGF-21, liver fibrosis and ultimately liver cancer.