The impact of hepatitis C virus NS5A protein mutations on interferon alfa (IFN-α) signaling pathway, cell proliferation, and viability is an important issue that is still under debate. We have therefore combined transient and stable expression in a human hepatocytic cell line (Huh7) of 3 full-length NS5A sequences, isolated from patients with or without response to IFN-α therapy. Expression of all 3 NS5A-reduced IFN-α global antiviral activity on both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) replication. We did not show, however, an effect of these 3 NS5A proteins on double-stranded RNA–dependent kinase (PKR) expression and activity as well as colocalization and coimmunoprecipitation between NS5A and PKR. We also failed to show an effect of the 3 NS5A mutants tested on cell proliferation and viability. Overall, our results support an important role of NS5A in controlling IFN-α antiviral activity; they show, however, that PKR-independent mechanisms are implicated, at least in liver-derived cells.