Cell Culture And Animal Models of HCV Infection And HCV-Host interactions

HCV对宿主机器的依赖性既复杂又广泛。这些宿主依赖性中的每一种都是潜在的治疗靶点。以前的努力已经成功地发现了HCV复制的重要步骤，但病毒生命周期中的许多基本过程仍然没有特征。使用基于RNAi的遗传学和感染性HCV细胞培养系统，我们进行了无偏倚的全基因组筛选，以确定生产性HCV感染所需的宿主因素。我们应用了两部分筛选方案来确定参与完整病毒生命周期的宿主因素，从病毒进入到感染性病毒的产生。随后进行了验证筛选，以尽量减少潜在的脱靶效应。在初始筛选中鉴定了512个基因，并通过验证测定确认了262个基因。我们确定了238个宿主易感因子（HSF）和24个宿主耐药因子（HRF），其中大多数以前与HCV无关。在这262个经验证的命中中，45个针对晚期病毒感染。这些宿主基因和其他已发表的数据库的综合生物信息学分析揭示了HCV对细胞过程和分子功能的广泛而复杂的依赖性，并且还涉及调节HCV感染的新细胞信号传导途径。包括TGF-β、ErbB、MAPK、粘着斑和泛素蛋白水解在内的几个关键通路在生物信息学分析中特别丰富。通过应用各种病毒学检测和分子技术，正在建立与HCV生命周期的每个步骤相关的细胞途径和机制的综合图谱，包括病毒进入，细胞内运输，病毒RNA复制和翻译，多蛋白加工，病毒粒子组装和分泌。HCV-宿主相互作用的全球识别和表征将显着推进我们对HCV相关发病机制的理解，从而阐明预防和治疗干预的潜在有价值的目标。 基于感染性HCV细胞培养系统，我们还与NIH化学基因组学中心合作建立了一种基于细胞的小分子化学文库高通量筛选（HTS）方法。NCGC收集了超过250，000种化合物，并建立了HTS设施。通过进行基于细胞的HTS，我们希望为HCV治疗开发确定新的靶点和先导化合物。 丙型肝炎病毒（HCV）株JFH-1的鉴定使感染性细胞培养系统的成功开发成为可能。虽然该毒株在细胞培养中能有效复制并产生感染性病毒，但其体内复制能力和致病机制尚不清楚。 我们以前报道过JFH-1病毒的体内表型。将细胞培养物产生的JFH-1病毒（JFH-1cc）和从中分离JFH-1的患者血清接种到黑猩猩中。 接种后3天，两只动物均变为HCV RNA阳性，但显示低水平病毒血症，无肝炎证据。 在JFH-1cc和患者血清感染的黑猩猩中，HCV病毒血症分别持续8周和34周。 免疫学分析显示，HCV特异性免疫反应在两种动物中诱导相似。 这项研究表明，HCV JFH-1株在黑猩猩中引起减毒感染和低致病性，并且能够通过赋予增强的复制表型的独特突变在体内适应。作为后续研究，我们进行了一个全面的分析先天和获得性免疫后，HCV再次暴露的两个黑猩猩从HCV-JFH 1感染恢复。我们观察到，在异源再激发后，HCV再感染的预防依赖于肝内先天性免疫应答和细胞免疫应答的激活。此外，我们的研究结果表明，血清中和抗体可能有助于控制病毒复制和传播后立即同源HCV再挑战。我们的结论是，对HCV再感染的保护性免疫是由先天性和适应性免疫反应的复杂网络精心策划的。

HCV dependencies on the host machinery are both intricate and extensive. Each of these host dependencies is a potential therapeutic target. Previous efforts have been successful in discovering important steps in HCV replication, yet many fundamental processes in the viral lifecycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we performed an unbiased genome-wide screen to identify host factors required for productive HCV infection. We applied a two-part screening protocol to identify host factors involved in the complete viral lifecycle, from viral entry to production of infectious virus. A validation screen was subsequently performed to minimize potential off-target effects. 512 genes were identified in the initial screen and 262 were confirmed by the validation assay. We identified 238 host susceptibility factors (HSFs) and 24 host resistance factors (HRFs), the majority of which were not previously linked to HCV. Of these 262 validated hits, 45 target late-stage viral infection. Integrative bioinformatics analyses of these host genes and other published database revealed a broad and complex dependency of HCV on cellular processes and molecular functions, and also implicated novel cellular signaling pathways modulating HCV infection. Several key pathways including TGF-beta, ErbB, MAPK, focal adhesion and ubiquitin proteolysis are particularly enriched in the bioinformatics analysis. By applying various virologic assays and molecular techniques, a comprehensive map of cellular pathways and machineries that are associated with each steps of HCV lifecycle, including viral entry, intracellular trafficking, viral RNA replication and translation, polyprotein processing, virion assembly and secretion, are being established. A global identification and characterization of HCV-host interactions will significantly advance our understanding of HCV-related pathogenesis, and hence illuminates potentially valuable targets for prophylactic and therapeutic interventions. Based on the infectious HCV cell culture system, we are also setting up a cell-based assay for high-throughput screening (HTS) of small molecule chemical library in collaboration with the NIH Chemical Genomics Center. The NCGC has a large collection of over 250,000 chemical compounds and has established the facility for HTS. By performing cell-based HTS, we hope to identify novel targets and lead compounds for HCV therapeutic development. The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. We previously reported the in vivo phenotype of the JFH-1 virus. Cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation, but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. This study shows that the HCV JFH-1 strain causes attenuated infection and low pathogenicity in chimpanzees, and is capable of adapting in vivo with a unique mutation conferring enhanced replicative phenotype. As a follow-up study, we performed a comprehensive analysis of the innate and adaptive immunity following HCV re-exposure of the two chimpanzees recovered from HCV-JFH1 infection. We observed that prevention of HCV re-infection upon heterologous re-challenge depend on both the activation of intrahepatic innate and cellular immune responses. Furthermore, our results suggest that serum neutralizing antibodies may contribute to the control of viral replication and spread immediately after homologous HCV re-challenges. We conclude that protective immunity against HCV re-infection is orchestrated by a complex network of innate and adaptive immune responses.