In vitro selection and characterization of HCV replicons resistant to multiple non-nucleoside polymerase inhibitors.

To delay or prevent the selection of HCV drug-resistant variants, combination therapy will be needed. Our aim was to determine the antiviral efficacy of various combinations of non-nucleoside polymerase inhibitors (NNI) (that have a different allosteric binding site) and the barrier towards resistance development of such combinations.Short-term antiviral combination assays were performed in a checkerboard format. Resistance selection experiments employing HCV replicons were performed using two different protocols: (i) a short-term treatment with fixed concentrations and (ii) a long-term treatment with increasing concentrations.All pair-wise combinations of NNI resulted in an additive antiviral effect in short-term antiviral assays. Combination treatment of two NNIs markedly reduced or even prevented the emergence of double resistant colonies. However, double and even triple NNI-resistant variants emerged readily when relatively low starting concentrations were used in a long-term selection protocol. Genotyping confirmed the presence of the previously published resistance mutations. For some NNI, different signature mutations appeared depending on the other NNI in the particular combination. Remarkably, variants that were selected to be resistant to three different classes of NNIs [a thiophene carboxylic acid (TCA), a benzimidazole (JT-16), and a benzofuran (HCV-796)] proved resistant to yet a fourth class of NNIs (benzothiadiazines).Double and even triple NNI-resistant HCV replicons can be readily selected with a stepwise resistance selection protocol. Depending on the particular combination, different signature mutations may arise for some NNI. Resistance to three classes of NNI resulted in resistance to yet a fourth class.