Identification and characterization of cellular factors involved in HCV entry

描述(由申请人提供)：丙型肝炎仍然是一个主要的全球公共卫生问题，尽管干扰素治疗取得了重大进展。新一代特定的抗病毒药物正在进入临床试验，但早期结果表明，即使在短期服用后，也确实出现了耐药变异，需要联合治疗才能有效地控制和根除病毒。到目前为止，大多数努力都集中在基因组RNA翻译或RNA复制所涉及的病毒靶标上。模拟完整的丙型肝炎病毒复制周期的高效细胞培养系统的出现，包括病毒粒子的组装、出口和进入，为基础和应用研究打开了新的机会。这项建议的重点是确定丙型肝炎病毒进入宿主细胞所需的细胞分子以及生产性进入所需的一系列事件。丙型肝炎病毒E2糖蛋白结合细胞表面分子，如Tetraspannin CD81和清道夫受体SR-BI参与了丙型肝炎病毒的进入，但它们既不足以进入，也不能确定它们的确切作用。我们调查了人类CD81+SR-B1+细胞系，发现了几个不能支持丙型肝炎病毒进入的细胞系。其中293T细胞被用来筛选一个新的可回收逆转录病毒文库，该文库是由丙型肝炎病毒允许的Huh-7.5细胞制成的。这一筛选确定了一种进入丙型肝炎病毒所需的新分子，Claudin-1(CLDN1)。CLDN1是一种跨膜的细胞表面蛋白，以前在紧密连接中发现。CLDN1在293T细胞中的表达使其对丙型肝炎病毒伪粒子(HCVpp)感染和细胞培养产生丙型肝炎病毒(HCVcc)完全允许。依赖CLDN1的丙型肝炎病毒进入不同的丙型肝炎病毒囊膜，需要CD81。CLDN1的表达与HCVpp进入靶细胞的能力有关。我们建议绘制丙型肝炎病毒进入所需的CLDN1的功能决定因素图，定义丙型肝炎病毒进入人和小鼠细胞所需的额外分子，并剖析这些分子在丙型肝炎病毒进入中的作用。这些研究将提供丙型肝炎病毒进入所需的细胞相互作用的详细图景，并对新的抗病毒方法和小动物模型的开发产生影响。

DESCRIPTION (provided by applicant): 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.