Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes UL30 and UL54, respectively, encoding the DNA polymerase (pol). In this study, we analyzed the impact of substitutions located in helix K and region II that are involved in the conformational changes of the DNA pol. Theoretical substitutions were identified by sequences alignment of the helix K and region II of human herpesviruses (susceptible to PFA) and bacteriophages (resistant to PFA) and introduced in viral genomes by recombinant phenotyping. We characterized the susceptibility of HSV-1 and HCMV mutants to PFA. In UL30, the substitutions I619K (helix K), V715S, and A719T (both in region II) increased mean PFA 50% effective concentrations (EC50s) by 2.5-, 5.6-, and 2.0-fold, respectively, compared to the wild type (WT). In UL54, the substitution Q579I (helix K) conferred hypersusceptibility to PFA (0.17-fold change), whereas the substitutions Q697P, V715S, and A719T (all in region II) increased mean PFA EC50s by 3.8-, 2.8- and 2.5-fold, respectively, compared to the WT. These results were confirmed by enzymatic assays using recombinant DNA pol harboring these substitutions. Three-dimensional modeling suggests that substitutions conferring resistance/hypersusceptibility to PFA located in helix K and region II of UL30 and UL54 DNA pol favor an open/closed conformation of these enzymes, resulting in a lower/higher drug affinity for the proteins. Thus, this study shows that both regions of UL30 and UL54 DNA pol are involved in the conformational changes of these proteins and can influence the susceptibility of both viruses to PFA.
Keywords: 3D modeling; DNA polymerase; HCMV; HSV-1; NH2-terminal domain; drug resistance mechanisms; foscarnet; herpes simplex virus; human cytomegalovirus; palm domain; resistance.