The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR 237-276 ) sequence associated with IFN resistance was not found, although the presence of Ala 245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a-and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.Hepatitis C virus (HCV) is a major cause of chronic liver disease leading to cirrhosis worldwide and is now recognized as a leading indication for orthotopic liver transplantation in the United States. The HCV genome has a high degree of genetic variability. Interindividual HCV sequence variability has led to the classification of at least six genotypes, while intraindividual variability is referred to as a quasispecies, which usually consists of a predominant viral variant and a variable mixture of highly genetically related yet distinct variants (23, 36).Interferon (IFN) monotherapy leads to sustained virological responses in less than 20% of chronic hepatitis C cases (47). The recent introduction of IFN plus ribavirin combination therapy has significantly improved response rates (8, 38). In multivariate analyses, HCV genotype and high viral load are two important virologic factors that independently predict the likelihood of treatment failure (37, 39)...