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: 7190151
• 负责人: Charles M Rice
• 金额: $ 42.25万
• 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/7190151
• 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

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.

描述（由申请人提供）：尽管基于干扰素的治疗取得了重大进展，但丙型肝炎仍然是一个主要的全球公共卫生问题。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，即使在短期给药后，也表明确实出现了耐药变异，需要联合治疗才能有效控制和根除病毒。迄今为止，大多数努力都集中在参与基因组RNA翻译或RNA复制的病毒靶标上。高效细胞培养系统的出现，模仿完整的HCV复制周期，包括病毒体组装，出口和进入，开辟了新的机会，基础和应用研究。该提案的重点是定义HCV进入宿主细胞所需的细胞分子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CD 81和清道夫受体SR-BI参与HCV进入，但它们既不足以进入，也没有明确的作用。我们调查了人CD 81 + SR-B1+细胞系，并确定了几个不能支持HCV进入。其中之一，293 T细胞，被用来筛选一种新的可回收的逆转录病毒cDNA文库从HCV-允许的Huh-7.5细胞。该筛选鉴定了HCV进入所需的新分子Claudin-1（CLDN 1）。CLDN 1是一种以前在紧密连接中发现的多跨膜细胞表面蛋白。CLDN 1在293 T细胞中的表达使得它们完全允许HCV假颗粒（HCVpp）和细胞培养物产生的HCV（HCVpp）的感染。对于不同的HCV包膜观察到CLDN 1依赖性HCV进入，并且需要CD 81。CLDN 1表达与HCVpp进入靶细胞的能力相关。我们建议映射CLDN 1的HCV进入所需的功能决定簇，定义HCV进入人类和小鼠细胞所需的其他分子，并剖析这些分子在HCV进入中的作用。这些研究将提供HCV进入所需的细胞相互作用的详细情况，并对开发新的抗病毒方法和小动物模型产生影响。