Identification of alpha interferon-induced envelope mutations of hepatitis C virus in vitro associated with increased viral fitness and interferon resistance.

Alpha interferon (IFN-alpha) is an essential component of innate antiviral immunity and of treatment regimens for chronic hepatitis C virus (HCV) infection. Resistance to IFN might be important for HCV persistence and failure of IFN-based therapies. Evidence for HCV genetic correlates of IFN resistance is limited. Experimental studies were hampered by lack of HCV culture systems. Using genotype (strain) 1a(H77) and 3a(S52) Core-NS2 JFH1-based recombinants, we aimed at identifying viral correlates of IFN-alpha resistance in vitro. Long-term culture with IFN-alpha 2b in Huh7.5 cells resulted in viral spread with acquisition of putative escape mutations in HCV structural and nonstructural proteins. Reverse genetic studies showed that primarily amino acid changes I348T in 1a(H77) E1 and F345V/V414A in 3a(S52) E1/E2 increased viral fitness. Single-cycle assays revealed that I348T and F345V/V414A enhanced viral entry and release, respectively. In assays allowing viral spread, these mutations conferred a level of IFN-alpha resistance exceeding the observed fitness effect. The identified mutations acted in a subtype-specific manner but were not found in genotype 1a and 3a patients, who failed IFN-alpha therapy. Studies with HCV recombinants with different degrees of culture adaptation confirmed the correlation between viral fitness and IFN-alpha resistance. In conclusion, in vitro escape experiments led to identification of HCV envelope mutations resulting in increased viral fitness and conferring IFN-alpha resistance. While we established a close link between viral fitness and IFN-alpha resistance, identified mutations acted via different mechanisms and appeared to be relatively specific to the infecting virus, possibly explaining difficulties in identifying signature mutations for IFN resistance.