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。 最近的功能基因组学研究，包括全基因组的小干扰RNA（siRNA）的屏幕表明，丙型肝炎病毒（HCV）利用广泛的网络的主机因素生产性感染和繁殖。这些增选的宿主功能如何与HCV复制周期的各个步骤相互作用，并对HCV感染产生促或抗病毒作用，在很大程度上仍不清楚。在这里，我们提出了一个公正的和系统的策略，从我们以前的感染性HCV（HCVRNA）siRNA屏幕上发现的功能询问HCV宿主的依赖性。应用功能基因组学方法和各种体外丙型肝炎病毒模型系统，包括丙型肝炎病毒假颗粒（HCVpp）、单循环感染性颗粒（HCVsc）、亚基因组复制子和丙型肝炎病毒细胞培养系统（HCVcc），我们鉴定并表征了每个步骤所需的新型宿主因子或途径。丙型肝炎病毒复制周期。特别是，我们发现了多种HCV进入因子，包括E-钙粘蛋白，胆碱激酶，NADPH氧化酶CYBA，Rho GTdR RAC 1和SMAD家族成员6。我们还证明了鸟嘌呤核苷酸结合蛋白GNB 2L 1、E2遍在蛋白缀合酶UBE 2 J1和39种其他宿主因子是HCV RNA复制所需的，而去遍在蛋白酶USP 11和多个其他细胞基因专门参与HCV IRES介导的翻译。还鉴定了靶向HCV复制或翻译的抗病毒因子家族。此外，各种病毒学分析证实，66个宿主因子参与HCV组装或分泌。这些基因包括胰岛素降解酶（IDE），一种前病毒因子，和N-Myc下调基因1（NDRG 1），一种抗病毒因子。我们的研究结果的生物信息学荟萃分析与以前发表的HCV宿主因子的文献挖掘相结合，可以构建一个广泛的细胞网络和完整的HCV复制周期所涉及的途径的路线图。这项对HCV宿主依赖性的全面研究为病毒感染、发病机制和潜在治疗靶点提供了新的见解。 使用相同的筛选技术，我们进行了一个公正的策略，以确定与HCV感染相关的细胞miRNA，并在功能上询问这些miRNA与我们以前的HCV小干扰RNA（siRNA）筛选数据库，以获得一个广泛的细胞/病毒调控网络在生产性HCV感染。我们通过使用两部分免疫染色格式进行了组合的全基因组miRNA（miRBase Sequence 13.0中的1000个miRNA）模拟抑制剂筛选。在初步筛选中，我们鉴定了100种减少（抗病毒）或增强（前病毒）HCV感染的miRNA。其中60个通过使用荧光素酶报告病毒的二次筛选进行验证。24个miRNAs是前病毒的，36个是抗病毒的。miR 122是筛选中确认的前病毒miRNA，另一种最近显示在HCV复制中起作用的miRNA miR 196也是确认的命中。通过使用各种HCV模型系统，这些新的miRNA中的大多数可以被分配到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细胞和原代人肝细胞中的总体miRNA表达分析显示，许多miRNA受到HCV感染的调控，其中一些也是上述全基因组功能筛选的验证命中，表明miRNA调控与HCV感染之间存在复杂的相互作用。我们进一步表征了两种经验证的miRNA对HCV传播的影响，并证明这些miRNA靶向我们的siRNA筛选中鉴定的某些宿主因子，这可能解释了这些miRNA对HCV感染的功能性影响。对调节HCV整个生命周期的细胞miRNAs的全面研究将对HCV的发病机制产生重要的见解，并提供新的治疗靶点。