Role of mitochondrial core protein in Hepatitis C Virus pathogenesis

线粒体核心蛋白在丙型肝炎病毒发病机制中的作用

• 批准号: 7197687
• 负责人: Melanie Maria Ott
• 金额: $ 9.75万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7197687
• 关键词: Aftercare; Amino Acid Motifs; Amino Acids; Apoptosis; Apoptosis Regulator; Apoptotic; Biological Assay; C-terminal; Carnitine Palmitoyltransferase I; Cell Fractionation; Cell physiology; Cells; Chronic Hepatitis; Cirrhosis; Condition; Core Protein; Cytoplasm; Data; Electrons; Endopeptidases; Enzymes; Family; Fatty Acids; Genome; Glycoproteins; Goals; Green Fluorescent Proteins; Hela Cells; Hepatic; Hepatitis B Virus; Hepatitis C; Hepatitis C virus; Hepatocyte; Human; Human papillomavirus 16 E1 protein; Immunoelectron Microscopy; Immunofluorescence Microscopy; In Vitro; Individual; Infection; Intracellular Membranes; Length; Lentivirus Vector; Lipids; Liver; Liver Cirrhosis; Localized; Location; Mitochondria; Molecular; Mus; Mutation; N-terminal; Nucleocapsid; Nucleotides; Outer Mitochondrial Membrane; Peptide Hydrolases; Peptide Signal Sequences; Point Mutation; Polyproteins; Primary carcinoma of the liver cells; Process; Property; Protein Precursors; Proteins; RNA; RNA Viruses; Rate; Replicon; Risk; Role; Series; Signal Transduction; Stimulus; Stretching; Surface; T-Lymphocyte; Testing; Transgenic Mice; Translations; Triglycerides; Viral; Viral Proteins; hepatitis C virus nucleocapsid protein; lipid metabolism; member; mutant; oral fibroblast; promoter; research study; response; uptake; vector; virus core; virus pathogenesis

DESCRIPTION: Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis worldwide. Approximately 170 million people are chronically infected and at risk to develop fatal liver damage, such as liver cirrhosis and hepatocellular carcinoma. The core protein represents the putative viral nucleocapsid and influences many cellular functions. Importantly, transgenic mice expressing core accumulate intracellular lipids, a condition called steatosis, and develop hepatocellular carcinoma. We observed that a fraction of core colocalized with mitochondrial markers in core-expressing HeLa and in Huh-7 cells containing the full-length HCV replicon. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that core is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C-terminus of processed core conferred mitochondrial localization when fused to green fluorescent protein. We seek to examine whether the presence of core at the surface of mitochondria directly influences mitochondrial functions. We will generate mutants in the mitochondrial targeting motif and examine their effect on cellular lipid accumulation and apoptosis. Our preliminary experiments show that wild type core markedly induced lipid droplet formation in Jurkat T cells. We further observed a strong proapoptotic effect of core in primary oral fibroblasts after treatment with apoptosis-inducing agents. The interaction of core with several candidate proteins located at the mitochondrial outer membrane will be examined. Potential core targets include carnitine palmitoyltransferase I, a rate-limiting enzyme involved in the uptake of fatty acids by mitochondria, and members of the Bcl-2 family of apoptosis regulators. We anticipate that our studies will help elucidate the mechanisms of how HCV core induces steatosis and hepatocellular carcinoma.

描述：丙型肝炎病毒 (HCV) 感染是全世界慢性肝炎的主要原因。大约 1.7 亿人受到慢性感染，并面临发生致命肝损伤的风险，例如肝硬化和肝细胞癌。核心蛋白代表假定的病毒核衣壳并影响许多细胞功能。重要的是，表达核心的转基因小鼠会积累细胞内脂质（一种称为脂肪变性的病症），并发展为肝细胞癌。我们观察到，在表达核心的 HeLa 和含有全长 HCV 复制子的 Huh-7 细胞中，一部分核心与线粒体标记共定位。使用免疫电子显微镜和体外线粒体输入测定，我们表明核心位于线粒体外膜上。当与绿色荧光蛋白融合时，加工核心的疏水性 C 末端内的一段 10 个氨基酸赋予线粒体定位。我们试图检查线粒体表面核心的存在是否直接影响线粒体功能。我们将在线粒体靶向基序中产生突变体，并检查它们对细胞脂质积累和细胞凋亡的影响。我们的初步实验表明，野生型核心显着诱导 Jurkat T 细胞中脂滴的形成。我们进一步观察到用凋亡诱导剂处理后，核心对原代口腔成纤维细胞具有强烈的促凋亡作用。将检查核心与位于线粒体外膜的几种候选蛋白质的相互作用。潜在的核心靶标包​​括肉毒碱棕榈酰转移酶 I（一种参与线粒体摄取脂肪酸的限速酶）以及凋亡调节因子 Bcl-2 家族的成员。我们预计我们的研究将有助于阐明 HCV 核心如何诱导脂肪变性和肝细胞癌的机制。