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

HCV 感染和 HCV-宿主相互作用的细胞培养和动物模型

• 批准号: 8148826
• 负责人: T. Jake Liang
• 金额: $ 51.97万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148826
• 关键词: Animal Model; Cell Culture Techniques; Hepatitis C

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.

丙型肝炎病毒对宿主机制的依赖既复杂又广泛。这些对宿主的依赖都是潜在的治疗靶点。以前的努力已经成功地发现了丙型肝炎病毒复制的重要步骤，但病毒生命周期中的许多基本过程仍未确定。使用基于RNAi的遗传学和感染性的丙型肝炎病毒细胞培养系统，我们进行了一项无偏见的全基因组筛查，以确定产生丙型肝炎病毒感染所需的宿主因素。我们应用了两部分筛选方案来确定从病毒进入到传染性病毒产生的整个病毒生命周期中涉及的宿主因素。随后进行了验证筛选，以最大限度地减少潜在的偏离目标的影响。在初步筛选中鉴定出512个基因，其中262个基因经验证分析得到确认。我们确定了238个宿主易感因子(HSF)和24个宿主抵抗因子(HRF)，其中大多数以前与丙型肝炎病毒无关。在这262次有效的命中中，45次针对晚期病毒感染。对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表明，丙型肝炎病毒对细胞过程和分子功能具有广泛而复杂的依赖性，并暗示了调控丙型肝炎病毒感染的新的细胞信号通路。在生物信息学分析中，包括转化生长因子-β、ErbB、MAPK、焦点黏附和泛素蛋白水解酶在内的几个关键途径尤其丰富。通过应用各种病毒学分析和分子技术，正在建立与丙型肝炎病毒生命周期的每个步骤相关的全面的细胞路径和机制图，包括病毒进入、细胞内转运、病毒RNA复制和翻译、多蛋白加工、病毒粒子组装和分泌。对丙型肝炎病毒-宿主相互作用的全球鉴定和表征将极大地促进我们对丙型肝炎相关发病机制的理解，从而为预防和治疗干预提供潜在的有价值的靶点。 在传染性丙型肝炎病毒细胞培养系统的基础上，我们还与美国国立卫生研究院化学基因组学中心合作，建立了一种基于细胞的高通量筛选小分子化学文库(HTS)的方法。NCGC拥有超过25万种化合物的大量收藏，并为HTS建立了设施。通过进行基于细胞的HTS，我们希望为丙型肝炎的治疗开发确定新的靶点和先导化合物。 丙型肝炎病毒JFH-1株的鉴定使感染性细胞培养系统的成功开发成为可能。虽然该菌株在细胞培养中能高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。我们之前报道了JFH-1病毒的体内表型。将细胞培养产生的JFH-1病毒(JFH-1CC)和从中分离到JFH-1的患者血清接种到黑猩猩体内。两只动物在接种后3天都呈丙型肝炎病毒RNA阳性，但表现出低水平的病毒血症，没有肝炎的证据。在JFH-1CC和患者血清感染的黑猩猩中，丙型肝炎病毒血症分别持续8周和34周。免疫学分析表明，在两种动物中都诱导了类似的丙型肝炎病毒特异性免疫反应。这项研究表明，丙型肝炎病毒JFH-1株对黑猩猩的感染减弱，致病性低，并能够在体内适应独特的突变，从而增强复制表型。作为后续研究，我们对两只从丙型肝炎病毒JFH1感染中恢复的黑猩猩进行了丙型肝炎病毒再暴露后先天免疫和获得性免疫的全面分析。我们观察到，防止丙型肝炎病毒在异种再攻击时再次感染依赖于肝脏内天然免疫反应和细胞免疫反应的激活。此外，我们的结果表明，血清中和抗体可能有助于控制病毒复制和在同源丙型肝炎病毒再次攻击后立即传播。我们得出结论，针对丙型肝炎病毒再次感染的保护性免疫是由一个复杂的先天和获得性免疫反应网络协调的。