Defined regions of hepatitis C virus (HCV) envelope 2 (E2), PePHD, and nonstructural 5A (NS5A) protein (PKR-binding domain) have been shown to interact with interferon alfa (IFN-alpha)-inducible double-stranded RNA-activated protein kinase (PKR) in vitro, suggesting a possible mechanism of HCV to evade antiviral effects of IFN-alpha. The clinical correlation between amino acid mutations within the E2 PePHD or the NS5A PKR-binding domain and response to antiviral treatment in HCV-3a-infected patients is unknown. Thirty-three patients infected with HCV-3a isolates were treated with IFN-alpha with or without ribavirin. The carboxyterminal half of E2 and of the NS5A gene were sequenced. Sixteen patients achieved a sustained virological response (SR), 6 patients an end-of-treatment response with relapse thereafter (ETR), and 11 patients were nonresponders (NR). Within the PePHD of the E2 protein 0.5 (range, 0-2) mutations were observed in SR patients, whereas the number of mutations in ETR or NR patients was 0.2 (0-1). Quasispecies analyses showed almost no heterogeneity. The mean number of mutations within the PKR-binding domain of the NS5A protein was 1.6 (range, 0-4) in SR patients, 1 (0-2) in ETR patients, and 1.6 (0-3) in NR patients. Patients with higher numbers of mutations within the E2 or NS5A region showed a trend towards lower pretreatment viremia. Phylogenetic and conformational analyses of E2 or NS5A sequences allowed no differentiation between sensitive and resistant isolates. However, mutations within the E2 PePHD in SR patients were frequent, and hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 were exclusively observed in sustained virological responders.