Role of mitochondrial core protein in Hepatitis C Virus pathogenesis

描述：丙型肝炎病毒（HCV）感染是全球慢性肝炎的主要原因。大约有1.7亿人是慢性感染者，有可能发生致命的肝损伤，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积累细胞内脂质，这种情况称为脂肪变性，并发展为肝细胞癌。我们观察到一部分核心与线粒体标记共定位在核心表达HeLa和Huh-7细胞含有全长HCV复制子。利用免疫电镜和体外线粒体输入试验，我们发现，核心位于线粒体外膜。当与绿色荧光蛋白融合时，加工核心的疏水C末端内的10个氨基酸的延伸赋予线粒体定位。我们试图研究是否存在的核心在线粒体表面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，并研究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，野生型核心显著诱导Jurkat T细胞中的脂滴形成。我们进一步观察到一个强大的核心在原代口腔成纤维细胞凋亡诱导剂治疗后的促凋亡作用。核心与位于线粒体外膜的几个候选蛋白质的相互作用将被检查。潜在的核心目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。我们期望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。

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.