Studies of HCV Infection And HCV-Host interactions

HCV 感染和 HCV-宿主相互作用的研究

基本信息

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
项目类别：
财政年份：
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

项目摘要

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。之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然未知。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，以确定有效 HCV 感染所需的宿主因子。我们应用了两部分筛选方案来识别整个病毒生命周期中涉及的宿主因素，从病毒进入到传染性病毒的产生。随后进行验证筛选，以尽量减少潜在的脱靶效应。在初始筛选中鉴定出 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 个宿主抵抗因子 (HRF)，其中大多数以前与 HCV 没有关联。在这 262 个经过验证的命中中，有 45 个针对晚期病毒感染。对这些宿主基因和其他已发表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，并且还暗示了调节 HCV 感染的新细胞信号传导途径。生物信息学分析特别丰富了几个关键通路，包括 TGF-β、ErbB、MAPK、粘着斑和泛素蛋白水解。通过应用各种病毒学测定和分子技术，与 HCV 生命周期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、病毒体组装和分泌，正在被绘制出来。已确立的。 HCV-宿主相互作用的全球识别和表征将显着促进我们对 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，包括全基因组小干扰 RNA (siRNA) 筛选，表明丙型肝炎病毒 (HCV) 利用广泛的宿主因子网络进行有效感染和传播。这些选定的宿主功能如何与 HCV 复制周期的各个步骤相互作用，并对 HCV 感染发挥促病毒或抗病毒作用，目前仍不清楚。在这里，我们提出了一种公正且系统的策略，以功能性地询问我们之前的感染性 HCV (HCVcc) siRNA 筛选中发现的 HCV 宿主依赖性。应用功能基因组学方法和各种体外 HCV 模型系统，包括 HCV 伪颗粒 (HCVpp)、单周期感染颗粒 (HCVsc)、亚基因组复制子和 HCV 细胞培养系统 (HCVcc)，我们鉴定并表征了所需的新宿主因子或途径HCV 复制周期的每个单独步骤。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 α、NADPH 氧化酶 CYBA、Rho GTPase RAC1 和 SMAD 家族成员 6。我们还证明了鸟嘌呤核苷酸结合蛋白 GNB2L1、E2 泛素结合酶 UBE2J1 和 39 HCV RNA 复制需要其他宿主因子，而去泛素化酶 USP11 和多种其他细胞因子基因特别参与 HCV IRES 介导的翻译。还鉴定了针对 HCV 复制或翻译的抗病毒因子家族。此外，各种病毒学检测验证了 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 (NDRG1)（一种抗病毒因子）。对我们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相结合，可以构建完整 HCV 复制周期中涉及的细胞网络和途径的广泛路线图。这项对 HCV 宿主依赖性的综合研究为病毒感染、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，我们执行了一种公正的策略来识别与 HCV 感染相关的细胞 miRNA，并使用我们之前的 HCV 小干扰 RNA (siRNA) 筛选数据库对这些 miRNA 进行功能性询问，以得出生产性 HCV 感染中广泛的细胞/病毒调控网络。我们使用两部分免疫染色格式进行了组合全基因组 miRNA（miRBase 序列 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 个可减少（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了验证。 24 个 miRNA 是前病毒的，36 个是抗病毒的。 miR122 是筛选中已确认的原病毒 miRNA，而另一种 miRNA miR196 最近显示在 HCV 复制中发挥作用，也被证实是命中的。通过使用各种 HCV 模型系统，大多数新型 miRNA 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全局miRNA表达分析表明，许多miRNA受到HCV感染的调节，其中一些也是上述全基因组功能筛选的验证命中，表明miRNA之间存在复杂的相互作用调节和HCV感染。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 靶向我们的 siRNA 筛选中确定的某些宿主因子，这可能解释了这些 miRNA 对 HCV 感染的功能影响。对调节整个 HCV 生命周期的细胞 miRNA 的全面研究将为 HCV 发病机制提供重要的见解并提供新的治疗靶点。