Browsing by Subject "RNA Helicases"
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Item Control of the Interferon Regulatory Factor - 3 Antiviral Pathway by The Hepatitis C Virus Ns3/4a Protease(2007-05-22) Foy, Eileen; Gale, Michael Jr.Hepatitis C Virus (HCV) is a major human pathogen that affects 200 million people worldwide. A majority of people exposed to HCV become chronically infected. In order to persist, the virus must encode mechanisms to subvert host immune defenses. We hypothesized that HCV disrupts critical host intracellular antiviral signaling pathways that culminate in activation of the antiviral response. Using the cell-based replicon system, HCV was found to inhibit the activation of critical intracellular signaling pathways. Our studies identified the NS3/4A protein as an interferon regulatory factor 3 (IRF-3) and NF-KB antagonist and further mapped this activity to the NS3/4A serine protease domain. HCV antagonism of IRF-3 and NF- KB prevents the expression of interferon (IFN) and IFN-stimulated genes required for viral clearance and the generation of a HCV-specific adaptive immune response. Upstream signaling components of IRF-3 were unknown, therefore, the NS3/4A protease was utilized as a tool to elucidate putative upstream signaling components. Characterization of Toll-like receptor 3 (TLR3) in HCV infection indicated that the NS3/4A protease cleaves TRIF, an essential adaptor protein required for TLR3 signaling. However the TLR3 pathway was not found to be essential for generating the intracellular antiviral response. Furthermore, these studies identified the retinoic acid inducible gene-I (RIG-I) as an intracellular viral double-stranded (ds)RNA sensor through molecular cloning and the characterization of a HCV replication permissive cell line defective in IRF-3 activation. The NS3/4A protease disrupted IRF-3 signaling through both the RIG-I pathway as well as the homologous MDA-5 pathway during viral infection. Lastly, these studies identified novel roles for TBK1, TANK and NEMO as signal transduction intermediates in the intracellular dsRNA response pathways. The HCV NS3/4A protease is critical for proper viral polyprotein processing, in addition to antagonism of intracellular antiviral signaling pathways. Therefore, the HCV NS3/4A protease is an ideal target for novel therapeutics. Indeed, the HCV replicon could be effectively eliminated from a persistently infected cell line through the use of an experimental NS3/4A protease inhibitor. These results offer great promise for the effective treatment of chronic HCV infection.Item Hepatitis C Virus Entry into Hepatocytes and Engagement of Innate Immune Defenses(2011-08-26T17:34:10Z) Owen, David Matthew; Gale, Michael Jr.Hepatitis C virus (HCV) infection is a major cause of liver disease and a global health problem with inadequate treatment options. An improved understanding of how HCV exploits and subverts host factors to establish infection should yield potential targets for therapy. This study uses a recently developed cell culture model of HCV infection to examine HCV entry and engagement of innate immune defenses. HCV associates with host apolipoproteins and enters hepatocytes through complex processes involving some combination of CD81, claudin-I, occludin, and scavenger receptor BI. Here I show that HCV forms a complex with very low density lipoprotein (VLDL) within infected hepatocytes and uses this association to support infection through the low density lipoprotein receptor (LDL-R). Blocking experiments demonstrate that beta-VLDL and apolipoprotein E (apoE) can compete with HCV for entry. Knockdown of the LDL-R by treatment with 25-hydroxycholesterol or siRNA ablated ligand uptake and reduced HCV infection of cells, whereas infection was rescued upon cell ectopic LDL-R expression. Analyses of gradient-fractionated HCV demonstrate that apoE is associated with HCV virions exhibiting peak infectivity and dependence upon the LDL-R for cell entry. These results define the LDL-R as a cooperative HCV co-receptor that supports viral entry and infectivity through interaction with apoE ligand present in an infectious HCV/lipoprotein complex comprising the virion. Furthermore, upon entry HCV induces an initial transient activation of interferon regulatory factor-3 (IRF3) which is dependent on retinoic acid inducible gene I (RIG-I) and interferon-beta promoter stimulator-1 (IPS-1). This activation produces an antiviral activity which inhibits HCV entry and replication. HCV NS3/4A protease activity blocks this activation within 48 hours. At later time points post infection HCV activates NF-kappaB in a RIG-I independent manner leading to inflammatory cytokine production. These studies identify 3 potential targets for future HCV therapy: 1) alteration of HCV-lipoprotein interaction to disrupt entry, 2) blockade of NS3/4A protease activity to restore innate antiviral response, and 3) modulation of HCV induced NF-kappaB signaling to downregulate chronic inflammation.