Studies of HCV Infection And HCV-Host interactions

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。 最近的功能基因组学研究，包括全基因组的小干扰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生产性感染和繁殖。这些被增选的宿主功能如何与丙型肝炎病毒复制周期的各个步骤相互作用，并在丙型肝炎病毒感染中发挥亲病毒或抗病毒作用，在很大程度上尚不清楚。在这里，我们提出了一种公正和系统的策略，从功能上询问从我们以前的感染性丙型肝炎病毒(HCVcc)siRNA筛查中发现的丙型肝炎病毒宿主依赖关系。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，包括丙型肝炎病毒伪粒子(HCVpp)、单周期感染颗粒(HCVsc)、亚基因组复制子和丙型肝炎病毒细胞培养系统(HCVcc)，我们鉴定和表征了丙型肝炎病毒复制周期每一步所需的新宿主因子或途径。特别是，我们发现了多种丙型肝炎病毒的进入因子，包括E-钙粘蛋白、胆碱激酶、NADPH氧化酶、Rho GTP酶RAC1和SMAD家族成员6。我们还发现了鸟嘌呤核苷酸结合蛋白GNB2L1、E2泛素结合酶UBE2J1等39个宿主因子是丙型肝炎病毒RNA复制所必需的，而去泛素化酶USP11和其他多种细胞基因特定地参与了丙型肝炎病毒IRES介导的翻译。针对丙型肝炎病毒复制或翻译的抗病毒因子家族也被确定。此外，各种病毒学分析证实，66个宿主因子参与了丙型肝炎病毒的组装或分泌。这些基因包括胰岛素降解酶(IDE)，一种前病毒因子，以及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丙型肝炎病毒宿主因子的文献挖掘相结合，可以构建完整的丙型肝炎病毒复制周期中涉及的细胞网络和路径的广泛路线图。这项对丙型肝炎病毒宿主依赖性的全面研究为病毒感染、发病机制和潜在的治疗靶点提供了新的见解。 使用相同的筛选技术，我们执行了一种无偏见的策略来识别与丙型肝炎病毒感染相关的细胞miRNAs，并用我们先前的丙型肝炎病毒小干扰RNA(SiRNA)筛查数据库对这些miRNAs进行功能询问，以得出在生产性丙型肝炎病毒感染中广泛的细胞/病毒调控网络。我们使用两部分免疫染色的形式进行了全基因组miRNA(miRBase序列13.0中的1000miRNA)模拟抑制物联合筛选。在初步筛选中，我们确定了100个降低(抗病毒)或增强(前病毒)丙型肝炎病毒感染的miRNAs。其中60个通过使用荧光素酶报告病毒的二级筛查进行了验证。24个miRNAs为前病毒，36个为抗病毒。MiR122是屏幕上确认的前病毒miRNA，另一种miRNA，miR196，最近被证明发挥了丙型肝炎病毒复制的作用，也是确认的热门。通过使用不同的丙型肝炎病毒模型系统，这些新的miRNAs中的大多数可以被分配到丙型肝炎病毒生命周期进入、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球miRNA表达分析表明，许多miRNAs受到丙型肝炎病毒感染的调控，其中一些也是上述全基因组功能筛选的验证结果，表明miRNA调控与丙型肝炎病毒感染之间存在复杂的相互作用。我们进一步鉴定了其中两个有效的miRNAs对丙型肝炎病毒传播的影响，并证明了这些miRNAs针对我们的siRNA筛选中确定的某些宿主因子，潜在地解释了这些miRNAs对丙型肝炎病毒感染的功能影响。对调控整个丙型肝炎病毒生命周期的细胞miRNAs的全面研究将为丙型肝炎病毒的发病机制提供关键的见解，并提供新的治疗靶点。

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.