Identification and characterization of cellular factors involved in HCV entry

参与 HCV 进入的细胞因子的鉴定和表征

• 批准号: 7329823
• 负责人: Charles M Rice
• 金额: $ 41.45万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7329823
• 关键词: Acids; Actins; Amino Acids; Animal Model; Animals; Antiviral Agents; Asialoglycoprotein Receptor; Binding; CD81 gene; Cell Culture System; Cell Line; Cell Surface Proteins; Cell physiology; Cell surface; Cells; Chimera organism; Clinical Trials; Collection; Combined Modality Therapy; Cultured Cells; Cytoskeleton; Development; Event; Facility Construction Funding Category; Family member; Generations; Genome; Genotype; Glycoproteins; Goals; Grant; Hepatitis C; Hepatitis C virus; Homo; Human; Infection; Interferons; Laboratories; Lipoproteins; Liver; Low Density Lipoprotein Receptor; Maps; Mediating; Membrane; Molecular; Molecular Cloning; Mus; Pan Genus; Pan troglodytes; Process; Proteins; Public Health; RNA; RNA replication; Reagent; Recycling; Replicon; Resistance; Retroviridae; Role; Serum; Small Interfering RNA; Surveys; Temperature; Tight Junctions; Time; Translations; Variant; Viral; Virion; Virus; Virus Replication; Xenograft Model; base; cDNA Library; claudin-1 protein; expression cloning; extracellular; hepatitis C virus envelope 2 protein; human PHEMX protein; novel; particle; permissiveness; physical property; research study; scavenger receptor; small hairpin RNA; tissue/cell culture; vector; virus envelope

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.

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- 以治疗为基础。新一代特定的抗病毒药物正在进入临床试验，但初步结果，即使在 短期给药，表明耐药变异确实出现，联合治疗将是 有效控制和根除病毒所需的。到目前为止，大多数努力都集中在涉及的病毒靶点上 在基因组RNA翻译或RNA复制中。高效的细胞培养系统的出现 完整的丙型肝炎病毒复制周期，包括病毒粒子组装、出口和进入，为 基础研究和应用研究。这项提案的重点是定义进入丙型肝炎病毒所需的细胞分子。 进入宿主细胞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 加入丙型肝炎病毒，但他们既不足以进入，也没有定义他们的确切作用。我们调查了 人CD81+SR-B1+细胞系，并鉴定出几个不能支持丙型肝炎病毒进入的细胞系。其中之一， 用293T细胞筛选到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个细胞。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 以前在紧密连接中发现的跨越细胞表面蛋白的多个膜。中的CLDN1表达式 293T细胞使其完全允许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)。观察依赖CLDN1的丙型肝炎病毒进入不同的丙型肝炎病毒包膜，并要求 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，定义了丙型肝炎病毒所需的额外分子 进入人类和小鼠细胞，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 提供进入丙型肝炎病毒所需的细胞相互作用的详细图片，并对 开发新的抗病毒方法和小动物模型。