Studies of HCV Infection And HCV-Host interactions

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

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. From the GW siRNA screen, we also identified a pivotal role of IKK-a in regulating cellular lipogenesis and HCV assembly. In this study, we defined and characterized NIK as an IKK-a 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-a activation, cellular lipid metabolism, and viral assembly. We demonstrated that NIK interacts with IKK-a to form a kinase complex in association with the stress granules, in which IKK-a 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-a, suggesting NIK acts upstream of IKK-a. 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. While therapy for HCV is highly effective, an effective prophylactic vaccine is still lacking due to the challenge of the highly diverse nature of the virus. HCV infection is mediated by its envelope glycoprotein E1 and E2 in the entry process with E2 binding to the cell membrane receptors and E1 possibly mediating endosomal fusion). The structure of E1E2 dimer has only been partially resolved by the crystallization of the core or ectodomain of E2 protein (cE2) and its complex with various neutralizing antibodies. Further structural understanding of the E1E2 heterodimers can advance the design of candidates for HCV vaccine development. By applying a well-developed cryo-electron microcopy/tomography (cryo-EM/ET) methods that include sample preparation (negative stain or vitrified frozen sample), data acquisition and data processing for both single particle and tomography available at the Frederick National Laboratory for Cancer Research, the Center for Molecular Microscopy (CMM), we studied the structure of hepatitis C virus (HCV) envelope glycoproteins E1E2 heterodimer with the aid of various well-defined monoclonal anti-E1 and E2 antibodies. The generation of recombinant full-length E1E2 heterodimers has been described previously and is currently being developed as an HCV vaccine candidate). Here we generated complexes of recombinant E1E2 heterodimer (genotype 1a, HCV1 clone) with various anti-HCV Fabs targeting specific E1 or E2 regions of the protein. Extensive negative-stain single particles datasets were collected on E1E2 heterodimer-Fab complexes. Despite the heterogenous nature of the particles from the raw EM images, we managed to generated 3D models after extensive 2D classification analysis. We then modeled the available crystal structures of the cE2 and Fab into the 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. On EM, the E1E2 heterodimer exists in monomeric form and consists of a main globular body presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the cE2 based on Fab binding. At current resolution of 30, our model clearly shows the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Further cryoEM work is being pursued to generate a more refined structural model of the E1E2 heterodimer. Such information would greatly facilitate HCV vaccine development.