Identification and characterization of cellular factors involved in HCV entry

丙型肝炎仍然是一个主要的全球公共卫生问题，尽管在干扰素- 基础治疗。新一代的特异性抗病毒药物正在进入临床试验，但早期结果，即使在 短期给药，表明耐药变异确实出现，联合治疗将 有效控制和根除病毒。迄今为止，大多数努力都集中在涉及的病毒靶点上 基因组RNA翻译或RNA复制。高效细胞培养系统的出现模仿了 完整的HCV复制周期，包括病毒体组装、外出和进入，为HCV感染开辟了新的机会。 基础和应用研究。该提案的重点是定义HCV进入所需的细胞分子 进入宿主细胞和生产性进入所需的事件序列。HCV E2糖蛋白结合 细胞表面分子如四跨膜蛋白CD81和清道夫受体SR-BI参与 HCV进入，但它们既不足以进入，也没有定义它们的精确角色。我们调查 人CD81 + SR-B1+细胞系，并鉴定了几种不能支持HCV进入的细胞。其中之一， 293T细胞，筛选了一个新的可回收的逆转录病毒cDNA文库， 7.5细胞该筛选鉴定了HCV进入所需的新分子Claudin-1（CLDN1）。CLDN1是一种 以前在紧密连接中发现的多跨膜细胞表面蛋白。CLDN1表达 293T细胞使其完全允许HCV假颗粒（HCVpp）感染， 产生HCV（HCV-HCV）。对于不同的HCV包膜观察到CLDN1依赖性HCV进入，并且需要 CD81。CLDN1表达与HCVpp进入靶细胞的能力相关。我们建议绘制 HCV进入所需的CLDN1的功能决定簇，定义了HCV进入所需的其他分子。 进入人类和小鼠细胞，并剖析这些分子在HCV进入的作用。这些研究将 提供了HCV进入所需的细胞相互作用的详细图片， 开发新的抗病毒方法和小动物模型。

Hepatitis C continues to be a major global public health problem despite significant advances in interferon- based treatment. A new generation of specific antivirals is entering clinical trials but early results, even after short-term administration, suggest that resistant variants do emerge and that combination therapy will be needed for effective virus control and eradication. Most efforts to date have focused on viral targets involved in genome RNA translation or RNA replication. The advent of efficient cell culture systems mimicking the complete HCV replication cycle, including virion assembly, egress and entry, opens up new opportunities for basic and applied studies. This proposal is focused on defining the cellular molecules required for HCV entry into host cells and the sequence of events required for productive entry. HCV E2 glycoprotein binding cellular cell surface molecules such as the tetraspannin CD81 and scavenger receptor SR-BI participate in HCV entry, but they are neither sufficient for entry nor have their precise roles been defined. We surveyed human CD81+ SR-B1+ cell lines and identified several that were unable to support HCV entry. One of these, 293T cells, was used to screen a novel recyclable retrovirus cDNA library made from HCV-permissive Huh- 7.5 cells. This screen identified a new molecule required for HCV entry, Claudin-1 (CLDN1). CLDN1 is a multiple membrane spanning cell surface protein previously found in tight junctions. CLDN1 expression in 293T cells renders them fully permissive for infection by HCV pseudoparticles (HCVpp) and cell culture produced HCV (HCVcc). CLDN1 dependent HCV entry is observed for diverse HCV envelopes and requires CD81. CLDN1 expression correlates with the ability of HCVpp to enter target cells. We propose to map the functional determinants of CLDN1 required for HCV entry, define additional molecules required for HCV entry into human and murine cells, and dissect the roles of these molecules in HCV entry. These studies will provide a detailed picture of the cellular interactions required for HCV entry with implications for the development of new antiviral approaches and small animal models.