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: 10000721
• 负责人: T. Jake Liang
• 金额: $ 124.67万
• 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/10000721
• 关键词: Affect; Animal Model; Antiviral Agents; Binding Sites; Bioinformatics; Biological Assay; Biology; Cell Culture System; Cells; Chronic Hepatitis C; Complex; Cultured Cells; Data; Dependence; Disease; Environment; Gene Expression; Genes; Genetic; Genetic Transcription; HNF4A gene; Hepatic; Hepatitis C; Hepatitis C virus; Hepatocyte; Human; Impairment; Infection; Infectious hepatitides; Integration Host Factors; Life Cycle Stages; Ligands; Link; Lipids; Liver; Malignant Neoplasms; Maps; Measures; Mediating; Messenger RNA; MicroRNAs; Modeling; Molecular; Morbidity - disease rate; Morphogenesis; Mus; Myelogenous; Pathogenesis; Pathway interactions; Patients; Pattern; Phosphotransferases; Plasmids; Process; Production; Protein-Serine-Threonine Kinases; Proteins; RNA Interference; RNA replication; Receptor-Interacting Serine/Threonine Protein Kinase 2; Repression; Role; SCID Mice; SRE-2 binding protein; Seeds; Small Interfering RNA; Tissues; Translations; Untranslated Regions; Very low density lipoprotein; Viral; Viral Genome; Viral Pathogenesis; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; base; chemokine; density; gain of function; genome-wide; in vivo; inhibitor/antagonist; insight; iodixanol; lipid biosynthesis; lipid metabolism; liver biopsy; loss of function; mortality; novel; overexpression; particle; protein expression; response; stress granule; therapeutic target; transcriptomics; viral RNA; virus host interaction

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.

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，我们确定了许多以前未被认识到的生产性HCV感染所需的宿主因子。 跨膜脂质相关蛋白TM 6SF 2是HCV调节LVP形成和HCV生命周期的宿主依赖性因子。HCV通过极低密度脂蛋白（VLDL）途径进行形态发生、成熟和分泌，并以脂病毒颗粒（LVP）形式循环。我们进行了TM 6SF 2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水平来检查培养肝细胞中的HCV感染。在用过表达tm 6sf 2序列的质粒转染的Huh 7.5.1细胞中，通过碘克沙醇梯度测定来评估分泌的LVP的密度。我们测量了来自慢性丙型肝炎（CHC）患者、HCV感染的人源化Alb-uPA/SCID小鼠的肝脏和HCV感染的Huh7.5.1细胞的肝活检组织中的TM 6SF 2表达模式。肝细胞中的TM 6SF 2耗竭降低了病毒RNA和感染性病毒颗粒的分泌，而不影响HCV基因组的复制、翻译或组装。TM 6SF 2的过表达降低了细胞内HCV RNA和感染性LVP的水平，并且相反地增加了它们在培养上清液中的水平。在HCV感染的细胞中，TM 6SF 2过表达增强了上清液中较低密度组分中感染性LVP的产生。HCV感染增加了培养细胞、小鼠人源化肝脏和CHC患者肝脏中的TM 6SF 2表达。在CHC肝活检组织中，TM 6SF 2 mRNA水平与HCV RNA水平呈正相关。SREBP-2似乎介导HCV诱导TM 6SF 2表达。TM 6SF 2是感染性LVP的成熟、脂化和分泌所必需的;而HCV反过来上调TM 6SF 2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HCV组装中的关键作用。在这项研究中，我们将NIK定义和表征为IKK介导的前病毒效应中的IKK上游丝氨酸/苏氨酸激酶，以及HCV利用这种先天途径发挥其优势的机制。我们通过功能丧失和功能获得的方法操纵Huh7.5.1细胞中的NIK表达，并检查对IKK激活、细胞脂质代谢和病毒组装的影响。我们证明，NIK与IKK-相互作用，形成与应激颗粒相关的激酶复合物，其中IKK-在HCV感染后被磷酸化。NIK的消耗显着减少胞质脂滴含量和受损的HCV颗粒的生产。NIK过表达增强了HCV的组装，而在IKK-缺失的细胞中，这一过程被废除，表明NIK在IKK-的上游起作用。在HCV感染的肝细胞、移植人肝细胞的Alb-uPA/Scid小鼠的肝组织和慢性丙型肝炎患者中，NIK丰度增加。NIK mRNA在3个UTR中含有miR-122种子序列结合位点。miR-122模拟物和发夹抑制剂直接影响NIK水平。在我们的肝脏模型中，HCV感染显著降低了miR-122水平。我们证明了HNF 4A，一种已知的pri-miR-122转录调节因子，被HCV感染下调。NIK代表miR-122的真正靶点，其转录通过减少HNF 4A表达被HCV下调。这种作用，连同HCV复制对miR-122的隔离，导致NIK表达的去抑制以解除脂质代谢。 细胞microRNA（miRNAs）通过直接作用于病毒基因组或间接靶向病毒相关的宿主因子来调节HCV感染。最近，我们通过全基因组miRNA功能筛选和转录组学分析生成了HCVmiRNA相互作用的综合图谱。许多以前未被识别的细胞miRNA被鉴定为参与HCV感染，包括miR-135 a，一种人类癌症相关的miRNA。在本研究中，我们研究了miR-135 a在调节HCV生命周期中的作用，并表明它优先增强病毒基因组复制。基于生物信息学的综合分析和随后的功能测定揭示了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（RIPK 2）、髓样分化初级反应88（MYD 88）和C-X-C基序趋化因子配体12（CXCL 12），它们是miR-135 a的真正靶点。这些基因已显示在RNA复制阶段抑制HCV感染。我们的数据表明，关键宿主限制因子的抑制介导了miR-135 a对HCV传播的前病毒作用。此外，在培养的肝细胞和人肝脏中，miR-135 a的肝脏丰度被HCV感染上调，可能介导更有利的病毒复制环境，并可能导致HCV诱导的肝脏恶性肿瘤。这些结果为HCV宿主相互作用提供了新的见解，并揭示了将miRNA生物学与HCV发病机制联系起来的分子途径。