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。利用基于RNAi的遗传学和具有感染性的丙型肝炎病毒细胞培养系统，我们确定了许多以前未被识别的宿主因素，这些因素是产生丙型肝炎病毒感染所必需的。 跨膜脂质相关蛋白TM6SF2是丙型肝炎病毒在调节LVP形成和丙型肝炎病毒生命周期中的宿主依赖因子。丙型肝炎病毒选择极低密度脂蛋白(VLDL)途径进行形态发生、成熟和分泌，并以脂病毒颗粒(LVEP)的形式循环。我们进行了TM6SF2功能丧失和功能获得试验，并通过分析病毒RNA和蛋白表达以及感染性LVP水平来检测培养肝细胞中的丙型肝炎病毒感染情况。用碘二醇梯度法检测高表达TM6SF2基因的Huh7.5.1细胞分泌LVP的密度。我们检测了慢性丙型肝炎(CHC)患者的肝活检组织、感染丙型肝炎病毒的人源化Alb-uPA/SCID小鼠的肝脏以及感染丙型肝炎病毒的Huh7.5.1细胞中TM6SF2的表达模式。肝细胞中TM6SF2的缺失减少了病毒RNA和感染性病毒颗粒的分泌，而不影响丙型肝炎病毒基因组的复制、翻译或组装。TM6SF2的过表达降低了细胞内丙型肝炎病毒RNA和感染性LVP的水平，并反过来增加了它们在培养上清液中的水平。在丙型肝炎病毒感染的细胞中，TM6SF2的过表达促进了低密度上清液中感染性LVP的产生。丙型肝炎病毒感染可增加培养细胞、人源化小鼠肝脏和CHC患者肝脏中TM6SF2的表达。CHC肝活检组织中TM6SF2mRNA水平与丙型肝炎病毒RNA水平呈正相关。SREBP-2似乎介导了丙型肝炎病毒诱导TM6SF2的表达。TM6SF2是感染性LVP成熟、脂化和分泌所必需的；而丙型肝炎病毒反过来上调TM6SF2的表达，以促进其生产性感染。 从GW siRNA筛选中，我们还确定了ikk-在调节细胞脂肪生成和丙型肝炎病毒组装中的关键作用。在这项研究中，我们定义和表征了NiK作为IKK-上游丝氨酸/苏氨酸激酶在IKK介导的前病毒效应中的作用，以及丙型肝炎病毒利用这一先天途径发挥优势的机制。我们通过功能丧失和功能获得的方法在Huh7.5.1细胞中操纵Nik的表达，并检测其对IKK激活、细胞脂质代谢和病毒组装的影响。我们证明了Nik与IKK-相互作用，形成一个与应激颗粒相关的激酶复合体，其中IKK-在丙型肝炎病毒感染时被磷酸化。NIK的耗竭显著减少了胞浆中的脂滴含量，并损害了丙型肝炎病毒颗粒的产生。NIK过表达促进了丙型肝炎病毒的组装，这一过程在缺乏IKK-的细胞中被取消，表明NIK作用于IKK-的上游。在丙型肝炎病毒感染的肝细胞、植入人肝细胞的Alb-uPA/SCID小鼠和慢性丙型肝炎患者的肝组织中，NIK的丰度增加。Nik基因在3个非编码区含有一个miR-122种子序列结合位点。MIR-122模拟和发夹抑制剂直接影响NIK水平。在我们的肝脏模型中，miR-122水平因丙型肝炎病毒感染而显著降低。我们证明了已知的pri-miR-122转录调节因子HNF4a被丙型肝炎病毒感染下调。NIK代表了miR-122的一个真正的靶点，其转录被丙型肝炎病毒通过减少HNF4a的表达而下调。这一效应，加上丙型肝炎病毒复制对miR-122的隔离，导致NIK表达下调，从而解除对脂质代谢的调节。 细胞microRNAs(MiRNAs)通过直接作用于病毒基因组或通过靶向病毒相关宿主因子间接调控丙型肝炎病毒感染。最近，我们通过全基因组的miRNA功能筛选和转录组学分析，生成了一个全面的HCVmiRNA相互作用图谱。许多以前不受重视的细胞miRNAs被发现与丙型肝炎病毒感染有关，包括miR-135a，一种与人类癌症相关的miRNA。在本研究中，我们研究了miR-135a在调节丙型肝炎病毒生命周期中的作用，并表明它优先促进病毒基因组的复制。基于生物信息学的综合分析和随后的功能分析表明，三种抗病毒宿主因子，包括受体相互作用丝氨酸/苏氨酸激酶2(RIPK2)、髓系分化初级反应88(MYD88)和C-X-C基序趋化因子配体12(CXCL12)是miR-135a的真正靶点。这些基因已被证明在RNA复制阶段抑制丙型肝炎病毒感染。我们的数据表明，抑制关键宿主限制因子介导了miR-135a对丙型肝炎病毒传播的前病毒效应。此外，在培养的肝细胞和人的肝脏中，miR-135a的肝脏丰度都因丙型肝炎病毒感染而上调，这可能为病毒复制提供了更有利的环境，并可能导致丙型肝炎病毒诱导的肝脏恶性肿瘤。这些结果提供了对HCV宿主相互作用的新见解，并揭示了将miRNA生物学与丙型肝炎病毒致病机制联系起来的分子途径。

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 life cycle remain uncharacterized. Using RNAi-based genetics and an infectious HCV cell culture system, we identified many previously unrecognized host factors required for productive HCV infection. The transmembrane, lipid-associated protein TM6SF2 is such a host dependency factor for HCV in modulating LVP formation and HCV life cycle. HCV co-opts the very low density lipoprotein (VLDL) pathway for morphogenesis, maturation and secretion, and circulates as lipoviroparticles (LVPs). We conducted both TM6SF2 loss-of-function and gain-of-function assays and examined HCV infection in cultured hepatocytes by analyzing viral RNA and protein expression and infectious LVP levels. The density of secreted LVPs was evaluated by iodixanol gradient assay in Huh7.5.1 cells transfected with a plasmid overexpressing tm6sf2 sequence. We measured TM6SF2 expression patterns in liver biopsies from chronic hepatitis C (CHC) patients, livers of HCV infected humanized Alb-uPA/SCID mice, and HCV-infected Huh7.5.1 cells. TM6SF2 depletion in hepatocytes decreased viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatant. In HCV-infected cells, TM6SF2 overexpression enhanced production of infectious LVPs in lower density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice and CHC patient livers. TM6SF2 mRNA levels correlate positively with HCV RNA levels in CHC liver biopsies. SREBP-2 appears to mediate the induction of TM6SF2 expression by HCV. TM6SF2 is requisite for maturation, lipidation and secretion of infectious LVPs; and HCV, in turn, up-regulates TM6SF2 expression to facilitate its productive infection. From the GW siRNA screen, we also identified a pivotal role of IKK- in regulating cellular lipogenesis and HCV assembly. In this study, we defined and characterized NIK as an IKK- upstream serine/threonine kinase in IKK-mediated proviral effects and the mechanism whereby HCV exploits this innate pathway to its advantage. We manipulated NIK expression in Huh7.5.1 cells through loss- and gain-of-function approaches and examined the effects on IKK- activation, cellular lipid metabolism, and viral assembly. We demonstrated that NIK interacts with IKK- to form a kinase complex in association with the stress granules, in which IKK- is phosphorylated upon HCV infection. Depletion of NIK significantly diminished cytosolic lipid droplet content and impaired HCV particle production. NIK overexpression enhanced HCV assembly and this process was abrogated in cells deprived of IKK-, suggesting NIK acts upstream of IKK-. NIK abundance was increased in HCV-infected hepatocytes, liver tissues from Alb-uPA/Scid mice engrafted with human hepatocytes, and chronic hepatitis C patients. NIK mRNA contains a miR-122 seed sequence binding site in the 3 UTR. MiR-122 mimic and hairpin inhibitor directly affected NIK levels. In our hepatic models, miR-122 levels were significantly reduced by HCV infection. We demonstrated that HNF4A, a known transcriptional regulator of pri-miR-122, was downregulated by HCV infection. NIK represents a bona fide target of miR-122 whose transcription is downregulated by HCV through reduced HNF4A expression. This effect, together with the sequestering of miR-122 by HCV replication, results in de-repression of NIK expression to deregulate lipid metabolism. Cellular microRNAs (miRNAs) have been shown to modulate HCV infection via directly acting on the viral genome or indirectly through targeting the virus-associated host factors. Recently we generated a comprehensive map of HCVmiRNA interactions through genome-wide miRNA functional screens and transcriptomics analyses. Many previously unappreciated cellular miRNAs were identified to be involved in HCV infection, including miR-135a, a human cancer-related miRNA. In the present study, we investigated the role of miR-135a in regulating HCV life cycle and showed that it preferentially enhances viral genome replication. Bioinformatics-based integrative analyses and subsequent functional assays revealed three antiviral host factors, including receptor interacting serine/threonine kinase 2 (RIPK2), myeloid differentiation primary response 88 (MYD88), and C-X-C motif chemokine ligand 12 (CXCL12), as bona fide targets of miR-135a. These genes have been shown to inhibit HCV infection at the RNA replication stage. Our data demonstrated that repression of key host restriction factors mediated the proviral effect of miR-135a on HCV propagation. In addition, miR-135a hepatic abundance is upregulated by HCV infection in both cultured hepatocytes and human liver, likely mediating a more favorable environment for viral replication and possibly contributing to HCV-induced liver malignancy. These results provide novel insights into HCVhost interactions and unveil molecular pathways linking miRNA biology to HCV pathogenesis.