Role of the inflammasome in hepatitis C virus pathogenesis

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DESCRIPTION (provided by applicant): The role of host lipid metabolism has attracted considerable attention in hepatitis C virus (HCV) life cycle. Altered host lipid metabolism is the cause of fatty-liver disease. There is a fundamental gap in understanding how HCV infection induces the proteolytic activation of sterol regulatory element binding proteins (SREBPs), the master regulator of fatty-liver disease, which is observed in patients with chronic HCV infection. The long- term goal of this research is to define the molecular mechanisms of altered lipid homeostasis that will better characterize the pathogenesis of HCV-mediated liver disease. The objective of this proposal is to identify the molecular mechanisms of activation of SREBPs by HCV. The central hypothesis of the application is that induction of the caspase-1 inflammasome complex in HCV-infected cells mediates the activation of SREBPs. The rationale for the proposed research is that, by understanding how HCV-mediated caspase-1 inflammasome induces the activation of SREBPs, their activation can be inhibited in new and innovative approaches to arrest the production of HCV and the progression of liver disease associated with HCV-infection. Thus, the proposed research is relevant to NIH mission that pertains to developing fundamental knowledge that will potentially help design the alternate strategies in the treatment of chronic hepatitis C infection. Supported by strong preliminary data, the proposal's main hypothesis will be tested under two specific aims: 1) Determine the mechanism of caspase-1 activation by HCV; 2) Define the mechanism of SREBPs proteolytic activation. Under the first aim, the role of liver-specific inflammasome complex in caspase-1 activation will be investigated using novel bimolecular fluorescence complementation (BiFC) assay, proteomics/mass spectroscopy, real-time RT-PCR, siRNA technology, confocal microscopy, and protein-protein interaction. Under the second aim, the role of the components of the inflammasome complex such as, NALP3, ASC, and caspase-1 on the regulation of SCAP, Insig, and COPII proteins will be investigated using the similar methods as described for aim 1. The activation of SREBPs will be investigated using western-blot, confocal microscopy, and cell-based reporter assays. The results from these studies will yield novel insights into mechanisms of altered lipid homeostasis, liver oncogenesis, insulin resistance, and obesity associated with chronic HCV. At the completion of these studies our work is collectively expected to establish the potential role of HCV in the activation of caspase-1 inflammasome complex and proteolytic activation of SREBPs through the interactions with chronic inflammatory processes. This work will also define the novel caspase-1 inflammasome complex induction in human hepatocytes. The proposed research is significant because it is expected to provide novel strategies for targeting the viral or cellular determinants to arrest the production of HCV as well as the progression of liver disease associated with HCV-infection. In addition, it is expected that the results will fundamentally advance the fields of liver inflammation and lipid metabolism mediated by HCV-infection.