Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype

To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-α and then transfecting the total RNA derived from the IFN-α-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-α (200 IU/ml). These cloned cells were further treated with IFN-α or IFN-β (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (αR series) possessing the IFN-α-resistant phenotype and four replicon cell lines (βR series) possessing the IFN-β-resistant phenotype. Furthermore, we obtained an additional replicon cell line (αRmix) possessing the IFN-β-resistant phenotype by two rounds of prolonged treatment with IFN-α and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the αR series were highly resistant to both IFN-α and IFN-β, although the αR series containing αRmix were only partially resistant to both IFN-α and IFN-β. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the βR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo.