Amino acid (aa) substitutions of core 70 and 91 and in the NS5A (nonstructural protein 5A) interferon sensitivity determining region (ISDR) as well as genetic polymorphisms in the host interleukin-28B (IL28B) locus affect the outcome of interferon (IFN)-based therapies for patients with chronic hepatitis C. The combination of these factors and the quasispecies nature of the virus complicate understanding of the underlying mechanism. Using infectious hepatitis C virus (HCV) genotype 1b clone HCV-KT9, we introduced substitutions at both core aa70 (Arg to Gln) and aa91 (Leu to Met). We also introduced four and nine ISDR aa substitutions into core mutant HCV-KT9. Using human hepatocyte chimeric mice with different IL28B genotypes, we examined the infectivity, replication ability, and susceptibility to IFN of these clones. Although aa substitutions in the ISDR significantly impaired infectivity and replication ability of the virus, core aa70 and 91 substitutions did not. The effect of IFN treatment was similar in core wild-type and mutant viruses. Interestingly, virus titer was significantly higher in mice with the favorable IL28B allele (rs8099917 TT and rs12979860 CC) in the transplanted hepatocytes than in mice with hepatocytes from rs8099917 TG and rs12979860 TT donors (P < 0.001). However, the effect of IFN was significantly greater, and intrahepatic expression levels of IFN-stimulated genes were significantly higher in mice with the favorable IL28B allele. Conclusion: Our data suggest that HCV replication levels and response to IFN are affected by human hepatocyte IL28B single-nucleotide polymorphism genotype and mutations in the ISDR. The mechanism underlying the clinically observed association of wild-type core protein in eradicationfavorable host cells should be investigated further. (HEPATOLOGY 2011;54:764-771) H ronic hepatitis C virus (HCV) infection is the leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. 1,2 Interferon (IFN) is an essential component of therapy for patients with chronic HCV infection, and the most effective currently available therapy is combination therapy with pegylated (PEG)-IFN and ribavirin (RBV). 3-5 Among HCV genotypes, genotype 1 is the most resistant to