Identifying determinants of HCV tropism.

描述（由申请人提供）：据估计，全世界有1.7亿人感染丙型肝炎病毒（HCV），并有发展为慢性肝炎或肝硬化的风险，后者通常导致肝细胞癌。目前还没有疫苗，获得许可的治疗方法与一般疗效和显著毒性有关。尽管这一全球性公共卫生问题迫在眉睫，但由于缺乏关键的实验模型，我们对HCV复制和发病机制的基本了解仍然很差。例如，在体外培养病毒和表达天然的融合性包膜糖蛋白的困难极大地限制了HCV的趋向性和进入性的研究。这些都是病毒生物学的关键方面，因为包膜病毒感染的宿主范围和发病机制在很大程度上取决于病毒包膜糖蛋白与细胞表面受体的选择性相互作用。HCV研究的一个主要目标是了解HCV如何靶向肝脏以及它通过什么机制进入宿主细胞。最近，该领域的一个重大突破是HCV包膜糖蛋白伪型逆转录病毒的发展，这种病毒特异性地介导原代肝细胞和某些其他人类细胞的感染。我们将使用这个新的实验系统来研究HCV进入靶细胞。自然发生的HCV包膜糖蛋白的改变可能预示了体内受体使用和靶细胞趋向性的差异。为了研究HCV细胞向性的范围，将测试含有临床HCV分离物包膜糖蛋白的假型进入相关原代细胞和细胞系的能力。一种功能性cDNA克隆方法将用于鉴定特异性介导HCV进入不同靶细胞的细胞表面受体。然而，这些受体可能是普遍表达的，HCV靶向不同类型的细胞可能是由另一种机制决定的。我们最近证明了L-SIGN和DC-SIGN是特异性的HCV捕获受体，我们将探索它们是否介导靶细胞的感染，从而确定HCV的趋向性。我们工作的主要目的是确定介导HCV趋向性的基本蛋白质相互作用，这将为HCV受体和包膜糖蛋白的详细结构/功能分析奠定基础。

DESCRIPTION (provided by applicant): It is estimated that 170 million people worldwide are infected with the Hepatitis C virus (HCV) and are at risk of developing chronic hepatitis or cirrhosis, the latter often leading to hepatocellular carcinoma. There is currently no vaccine and licensed therapies are associated with modest efficacies and significant toxicities. Despite the urgency of this worldwide public health problem, our basic understanding of HCV replication and pathogenesis remains poor due to a lack of key experimental models. For example, difficulties in culturing the virus in vitro and expressing native, fusogenic envelope glycoproteins have greatly limited studies of HCV tropism and entry. These are critical aspects of viral biology because the host range and pathogenesis of enveloped virus infection is largely determined by the selective interaction of viral envelope glycoproteins with cell-surface receptors. A major goal in HCV research is to understand how HCV targets the liver and by what mechanism it enters host cells. Recently, a major breakthrough in the field has been the development of retroviruses pseudotyped with HCV envelope glycoproteins that specifically mediate infection of primary hepatocytes, as well as certain other human cells. We will use this new experimental system to study HCV entry into target cells. Alterations in naturally occurring HCV envelope glycoproteins may predicate differences in receptor usage and target cell tropism in vivo. To investigate the range of HCV cellular tropism, pseudotypes incorporating envelope glycoproteins from clinical HCV isolates will be tested for their ability to enter relevant primary cells and cell lines. A functional cDNA cloning approach will be used to identify cell-surface receptors that specifically mediate HCV entry into different target cells. However, these receptors may be ubiquitously expressed and HCV targeting to different cell types may be determined by another mechanism. We recently demonstrated that L-SIGN and DC-SIGN are specific HCV-capture receptors and we will explore whether they mediate infection of target cells in trans, thereby determining HCV tropism. The major objective of our work is to identify the basic protein interactions that mediate HCV tropism, which will serve as a foundation for detailed structure/function analyses of HCV receptors and envelope glycoproteins.