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.
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