Interferon alpha (IFN-alpha) inhibits hepatitis C virus (HCV) replication in vivo and in cell cultures by one or several mechanisms that are not yet understood. We sought to identify the viral targets of the IFN-alpha-induced cellular antiviral program in Huh7 cells expressing HCV subgenomic replicons. Our results revealed a tight linkage between translation, assembly of replication complexes and viral RNA synthesis, and indicated that the stability of amplified plus strand RNA was reduced in the presence of the cytokine. Moreover, HCV internal ribosomal entry site (IRES)-directed translation was inhibited approximately 2-fold in IFN-treated cells. In contrast, the synthesis of viral RNA did not seem to be directly affected by the antiviral program induced by the cytokine. Our results were consistent with a model predicting that the IFN-alpha-induced antiviral program could inhibit multiple steps of the HCV replication cycle, leading to a reduction in viral protein synthesis and eventually inhibition of viral RNA amplification.