Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Consejo Superior de Investigaciones Científicas (CSIC), Campus de Cantoblanco, 28049, Madrid, Spain.
Cell culture produced hepatitis C virus (HCV) has been subjected to up to 100 serial passages in human hepatoma cells in the absence or presence of different doses of interferon-α (IFN-α). Virus survival, genetic changes, fitness levels and phenotypic traits have been examined. While high initial IFN-α doses (increasing from 1 to 4 IU/ml) did not allow HCV survival beyond passage 40, a gradual exposure (from 0.25 to 10 IU/ml) allowed the virus to survive for at least 100 passages. The virus passaged in the presence of IFN-α acquired IFN-α resistance as evidenced by enhanced progeny production and viral protein expression in an IFN-α environment. A partial IFN-α resistance was also noted in populations passaged in the absence of IFN-α. All lineages acquired adaptative mutations, and multiple, non-synonymous mutations scattered throughout the genome were present in IFN-α-selected populations. Comparison of consensus sequences indicates a dominance of synonymous versus non-synonymous substitutions. IFN-α-resistant populations displayed decreased sensitivity to a combination of IFN-α and ribavirin. A phenotypic trait common to all assayed viral populations is the ability to increase shut-off of host cell protein synthesis, accentuated in infections with IFN-α-selected populations carried out in the presence of IFN-α. The trait was associated with enhanced phosphorylation of PKR and eIF2α, although other contributing factors are likely. The results suggest that multiple, independent mutational pathways can confer IFN-α resistance to HCV, and might explain why no unified picture has been obtained regarding IFN-α resistance in vivo.