A single drug-resistance mutation in HSV-1 UL52 primase points to a difference between two helicase-primase inhibitors in their mode of interaction with the antiviral target.

OBJECTIVES: To investigate the mechanism of action of the helicase-primase inhibitors (HPIs) BAY 57-1293 and BILS 22 BS by selection and characterization of drug-resistant herpes simplex virus (HSV)-1 mutants. METHODS: HSV-1 mutants were selected using BAY 57-1293 in Vero cells. Resistance mutations identified in the UL5 helicase or UL52 primase genes were validated by marker transfer. Cross-resistance to the structurally distinct BILS 22 BS was measured by ID(50) determinations. RESULTS: (i) A single mutation (UL52: A899T) confers 43-fold resistance to BAY 57-1293, but does not confer any resistance to BILS 22 BS. (ii) A double mutant (UL52: A899T and UL5: K356T) is 2500-fold resistant to BAY 57-1293, which is more than 17 times the sum of fold-resistance due to the individual mutations, UL52: A899T (43-fold) and UL5: K356T (100-fold). (iii) Virus containing the single helicase mutation and the double mutant with mutations in both helicase and primase showed equal resistance to BILS 22 BS (70-fold). CONCLUSIONS: By measuring the relative inhibitory concentrations required to overcome particular mutations in the helicase and primase proteins, evidence was obtained that BAY 57-1293 interacts with both components of the helicase-primase complex to achieve maximum potency, whereas for BILS 22BS, this may not be the case. Furthermore, our observations suggest that BAY 57-1293 interacts simultaneously with UL5 and UL52. Overall, the results suggest that these two potent HPIs interact differently with the helicase-primase complex.

High frequency of spontaneous helicase-primase inhibitor (BAY 57-1293) drug-resistant variants in certain laboratory isolates of HSV-1.

Herpes simplex virus (HSV) helicase-primase is the target for a new group of potent antivirals that show great promise in vivo. A claimed advantage of this class of compounds is the low rate of drug resistance, which is reported to occur at a lesser rate than acyclovir (ACV)-resistance in cell culture. We confirmed that BAY 57-1293 is highly active against HSV-1 and superior to ACV when tested in Vero cells. Notably, drug resistance was detected in laboratory working stocks in two different strains of HSV at 10(-4) to 10(-5) and there was evidence that the resistant variants were present in the virus population before the selection was applied. Plaque-purified clones obtained from the parental viruses showed a lower level of resistance selection in the presence of drug (10-6) and this value is similar to published reports. In the case of HSV-1 SC16, no difference was observed between a working stock and a plaque-pure clone in the rate of resistance to the nucleoside analogue ACV. The working stocks were found to contain variants with resistance to BAY 57-1293 ranging from approximately 15-fold to 4,000-fold suggesting that these viruses have the potential to subvert effective therapy. Sequence analysis of HSV-1 helicase protein showed that most of the amino acid substitutions in the variants described in this study tallied with published results, with some interesting exceptions in the case of HSV-1 strain PDK. Resistant variants did not readily revert to a sensitive phenotype in the absence of the inhibitor and representative BAY 57-1293-resistant variants were cross-resistant to an alternative helicase-primase inhibitor, BILS 22 BS. Variants resistant to BAY 57-1293 retained sensitivity to the nucleoside analogue, ACV.