Hepatitis C Virus NS3/4A Protease and the Intracellular Antiviral Response: Mapping Complex Virus-Host Interactions

Virus infection triggers an innate immune response characterized by host cell production of interferon (IFN). Intermediates of viral replication, including dsRNA, initiate a signaling cascade that is amplified within the cell and alerts neighboring cells of viral invaders. Recognition of dsRNA intermediates occurs through retinoic acid inducible gene-I (RIG-I). RIG-I elicits an antiviral state by binding to the IFN-beta Promoter Stimulator-1 (IPS-1) adaptor protein, activating the latent downstream transcription factors IRF-3 and NF-kappaB. These transcription factors bind to the promoter region of effector genes including IFN-beta, producing an antiviral amplification loop within and around the infected cell. This response is critical for immunity to infection. Hepatitis C virus (HCV) is a serious global health problem with 170 million people chronically infected. HCV persistence is linked to viral regulation of innate host defenses by the nonstructural 3/4A protein complex (NS3/4A) cleavage of IPS-1. NS3 structural composition includes an amino-terminal serine protease and a carboxy-terminal RNA helicase. A structure-function analysis of NS3/4A truncation and deletion mutations was conducted. Mutants lacking the helicase domain retained the ability to control RIG-I signaling, but this regulation was abrogated by truncation of the protease domain. Furthermore, treatment of HCV-infected cells with a NS3/4A protease inhibitor prevented IPS-1 proteolysis, restored RIG-I signaling, and decreased viral protein levels. These results indicate that the NS3/4A protease domain alone can target IPS-1 on the mitochondrial membrane. Current dogma holds that NS3/4A is located on the endoplasmic reticulum, thus the mechanism of NS3/4A targeting IPS-1, a mitochondrial membrane protein, remains unexplained. We have shown that NS3/4A distributes on mitochondria independently of the previously identified NS4A membrane localization motif, in a manner dependent on the first twenty amino acids of the NS3 protease domain. The functional domains of IPS-1 that direct the immune response have not been elucidated. We conducted a structure-function study of IPS-1 that revealed distinct processes of IRF-3 and NF-kappaB activation. Mutational analyses further identified areas of IPS-1 critical for mitochondrial localization, dimerization, and uncoupling IRF-3 and NF-kappaB signaling. These findings improve our understanding of IPS-1 function in innate immunity to virus infection.