Role of mitochondrial core protein in Hepatitis C Virus pathogenesis

丙型肝炎病毒(HCV)感染是全球慢性肝炎的主要原因。约有1.7亿人患有慢性感染，并面临发生致命肝损伤的风险，如肝硬变和肝细胞癌。核心蛋白代表假定的病毒核衣壳，影响许多细胞功能。重要的是，表达CORE的转基因小鼠会积聚细胞内的脂肪，这种情况被称为脂肪变性，并发展为肝细胞癌。我们观察到，在表达核心的HeLa细胞和含有全长丙型肝炎病毒复制子的Huh-7细胞中，部分核心与线粒体标记共存。利用免疫电子显微镜和体外线粒体导入实验，我们发现核心位于线粒体外膜上。当与绿色荧光蛋白融合时，加工核心疏水C末端的10个氨基酸的拉伸可以提供线粒体定位。我们试图研究线粒体表面核心的存在是否直接影响线粒体的功能。我们将在线粒体靶向基序中产生突变，并检测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细胞形成脂滴。我们进一步观察到，在原代口腔成纤维细胞中，CORE在用凋亡诱导剂处理后具有强烈的促凋亡作用。核心与位于线粒体外膜的几个候选蛋白的相互作用将被检测。潜在的核心靶点包括肉碱棕榈酰基转移酶I，一种参与线粒体吸收脂肪酸的限速酶，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丙型肝炎病毒核心是如何导致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.