Control of the Interferon Regulatory Factor - 3 Antiviral Pathway by The Hepatitis C Virus Ns3/4a Protease
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-<small>K</small>B antagonist and further mapped this activity to the NS3/4A serine protease domain. HCV antagonism of IRF-3 and NF- <small>K</small>B 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.