Studies of HCV Infection And HCV-Host interactions

HCV 感染和 HCV-宿主相互作用的研究

• 批准号: 8939616
• 负责人: T. Jake Liang
• 金额: $ 88.38万
• 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/8939616
• 关键词: Animal Model; Antiviral Agents; Bioinformatics; Biological Assay; Biological Models; Cell Culture System; Cell Culture Techniques; Cell physiology; Cells; Choline Kinase; Complex; Databases; Dependency; Deubiquitinating Enzyme; E-Cadherin; Eligibility Determination; Family; Family member; Focal Adhesions; Gene Expression; Genes; Genetic; Hepatitis C; Hepatitis C virus; Hepatocyte; Host resistance; Human; In Vitro; Individual; Infection; Infectious hepatitides; Insulinase; Integration Host Factors; Internal Ribosome Entry Site; Investigation; Life Cycle Stages; Link; Luciferases; MAP Kinase Gene; Maps; Mediating; Meta-Analysis; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; NADPH Oxidase; Pathogenesis; Pathway interactions; Play; Polyproteins; Predisposition; Process; Production; Proteolysis; Published Database; Publishing; RNA Interference; RNA replication; Regulation; Replicon; Reporter; Role; Signal Pathway; Small Interfering RNA; Staging; Techniques; Technology; Therapeutic Intervention; Transforming Growth Factor beta; Translations; Ubiquitin; Ubiquitin-Conjugating Enzymes; Validation; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; base; functional genomics; genome wide association study; genome-wide; guanine nucleotide binding protein; improved; in vivo; inhibitor/antagonist; insight; mortality; new therapeutic target; novel; particle; prophylactic; resistance factors; rho GTP-Binding Proteins; screening; text searching; therapeutic target; trafficking; viral RNA; virus host interaction; virus pathogenesis

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. Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets. Using the same screening technology, we performed an unbiased strategy to identify cellular miRNAs associated with HCV infection and functionally interrogate these miRNAs with our previous HCV small interference RNA (siRNA) screen database to derive an extensive cellular/viral regulatory network in productive HCV infection. We performed a combined genome-wide miRNA (1000 miRNA in miRBase Sequence 13.0) mimic-inhibitor screen by using a two-part immunostaining format. In the primary screen, we identified 100 miRNAs that either reduced (antiviral) or enhanced (proviral) HCV infection. 60 of them were validated by a secondary screen using a luciferase reporter virus. 24 miRNAs were proviral and 36 antiviral. miR122 was a confirmed proviral miRNA in the screen and one other miRNA, miR196, recently shown to play a role HCV replication, was also a confirmed hit. By using various HCV model systems, the majority of these novel miRNAs can be assigned to different stages of HCV life cycle entry, IRES-mediated translation, viral RNA replication, and assembly/release. In addition, our global miRNA expression analyses in both Huh7.5.1 cells and primary human hepatocytes revealed that many miRNAs are regulated by HCV infection and some of them are also validated hits of the above genome-wide functional screen, suggesting a complicated interaction between miRNA regulation and HCV infection. We further characterized two of the validated miRNAs for their effects on HCV propagation and demonstrated that these miRNAs target certain host factors identified in our siRNA screen, potentially explaining the functional effects of these miRNAs on HCV infection. A comprehensive investigation of cellular miRNAs modulating the complete HCV life cycle will yield critical insights into HCV pathogenesis and provide novel therapeutic targets.

HCV对主机机械的依赖性既复杂又广泛。这些宿主依赖性中的每一个都是潜在的治疗靶标。以前的努力已经成功地发现了HCV复制的重要步骤，但是病毒生命周期中的许多基本过程仍然没有表现。使用基于RNAi的遗传学和感染性HCV细胞培养系统，我们进行了无偏基因组筛查的筛查，以识别生产性HCV感染所需的宿主因素。我们应用了两部分筛选方案，以确定从病毒入口到传染病的产生的完全病毒生命周期涉及的宿主因素。随后进行验证屏幕以最大程度地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个宿主敏感性因子（HSF）和24个宿主抗性因子（HRF），其中大多数以前与HCV无关。在这262个经过验证的命中中，有45个目标后期病毒感染。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，还暗示了调节HCV感染的新型细胞信号通路。在生物信息学分析中特别丰富了包括TGF-β，ERBB，MAPK，局灶性粘附和泛素蛋白水解在内的几种关键途径。通过应用各种病毒学测定和分子技术，正在建立与HCV生命周期相关的每个步骤相关的细胞途径和机器的综合图，包括病毒输入，细胞内运输，病毒RNA复制和翻译，多蛋白质处理，多蛋白处理，多蛋白处理，Virion Assembly和分泌。 HCV-host相互作用的全球鉴定和表征将显着提高我们对HCV相关发病机理的理解，从而阐明潜在的预防性和治疗干预措施的潜在宝贵目标。 最近的功能性基因组学研究（包括全基因组小干扰RNA（siRNA）筛查）表明，丙型肝炎病毒（HCV）利用了广泛的宿主因素网络，用于生产性感染和传播。这些配合的宿主功能如何与HCV复制周期的各个步骤相互作用，并对HCV感染发挥促或抗病毒作用仍然在很大程度上不确定。在这里，我们提出了一种无偏的系统策略，可以在功能上询问从我们以前的感染性HCV（HCVCC）siRNA屏幕中发现的HCV宿主依赖性。应用功能性基因组学方法和各种体外HCV模型系统，包括HCV伪颗粒（HCVPP），单周期感染性颗粒（HCVSC），亚基因组复制子和HCV细胞培养系统（HCVCC），我们确定并表征了每个单独的步进循环的新型宿主或途径。 Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the去泛素化酶USP11和多个其他细胞基因特别参与HCV IRES介导的翻译。还确定了针对HCV复制或翻译的抗病毒因子家族。此外，各种病毒学测定法证明了HCV组装或分泌涉及66个宿主因素。这些基因包括胰岛素降解酶（IDE），前病毒因子和n-myc降低的基因1（NDRG1），一种抗病毒因子。我们的结果的生物信息学荟萃分析与先前发表的HCV宿主因子的文献挖掘整合在一起，允许在完整的HCV复制周期中构建广泛的蜂窝网络和途径的路线图。这项对HCV宿主依赖性的全面研究可产生对病毒感染，发病机理和潜在治疗靶标的新颖见解。 使用相同的筛选技术，我们执行了一种无偏的策略，以识别与HCV感染相关的细胞miRNA，并在功能上与以前的HCV小型干扰RNA（SIRNA）筛选数据库询问这些miRNA，以推导出广泛的蜂窝/病毒调节网络在生产性HCV感染中。我们使用两部分的免疫染色格式进行了合并的全基因组miRNA（Mirbase序列13.0中的1000 miRNA）。在主要屏幕中，我们确定了100个miRNA，可减少（抗病毒）或增强（前病毒）HCV感染。其中60个使用荧光素酶报告基因病毒通过二级筛选进行了验证。 24个miRNA是前病毒和36抗病毒。 MiR122是屏幕上确认的病毒miRNA，另一个miRNA MiRNA最近证明扮演了HCV复制的角色，也是确认的命中。通过使用各种HCV模型系统，这些新型miRNA的大多数可以分配给HCV生命周期进入，IRES介导的翻译，病毒RNA复制和组装/释放的不同阶段。此外，我们在HUH7.5.1细胞和原发性人肝细胞中进行的全局miRNA表达分析表明，许多miRNA受HCV感染的调节，其中一些受到了上述全基因组功能筛查的验证，这表明miRNA调节和HCV感染之间存在复杂的相互作用。我们进一步表征了两个经过验证的miRNA对HCV传播的影响，并证明这些miRNA靶向我们siRNA筛查中鉴定的某些宿主因子，这可能解释了这些miRNA对HCV感染的功能效应。对调节完整HCV生命周期的细胞miRNA的全面研究将产生对HCV发病机理的关键见解，并提供新颖的治疗靶标。