Viral and Host Genetic Determinants of Hepatitis C Virus Persistence and Interferon Resistance
Sumpter, Rhea Myers, Jr.
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Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), which is an important cause of cirrhosis and hepatocellular carcinoma. HCV replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. The development of the HCV RNA replicon system has allowed the study of persistent HCV RNA replication in tissue culture. We evaluated HCV/IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV genotype 1b subgenomic RNA replicon that differed only at two sites within the NS5A coding region. A replicon with a lysine (K) insertion at HCV codon 2040 (K2040) replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an leucine (L) to serine (S) point mutation at HCV codon 2198 (L2198S) replicated poorly and triggered a cellular response characterized by IFN-! production and low-level interferon-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high passage (HP) variant with 6 additional point mutations throughout the HCV protein-coding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-" treatment and was associated with viral-directed reduction in host cell expression and action of ISG56, an antagonist of HCV RNA translation. We also demonstrated that HCV subgenomic RNA replicons can be used to model the early events of HCV infection. We found that HCV RNA replicons rapidly induce the cellular antiviral response upon their transfection into host Huh7 cells and we determined that intracellular HCV double stranded RNA (dsRNA) is a potent agonist of host dsRNAactivated pathways. A Huh7 derived cell line that is highly permissive for transduction by HCV replicons is specifically defective in the activation of interferon regulatory factor (IRF)-3 by virus infection or HCV dsRNA transfection. We found that a mutation in the caspase recruitment domain (CARD) of the DExH/D-box helicase protein RIG-I, a component of the TLR3-independent intracellular dsRNA-responsive IRF-3 activation pathway, was responsible for this phenotype. Restoration of RIG-I-mediated IRF-3 activation through genetic complementation resulted in decreased permissiveness to HCV RNA replication. These results establish the RIG-I!IRF-3 pathway as a critical determinant of HCV persistence.