Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme

Abstract

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants were previously determined by restriction endonuclease analysis. These recombinants and their parental strains were employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to 3 inhibitors of DNA polymerase. The viral DNA polymerases induced by 2 recombinants and their parental strains were purified and shown to possess similar MW (142,000-144,000) and similar sensitivity to compounds which distinguish viral and cellular [hamster kidney BHK and African green monkey kidney vero] DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate and the 2'',3''-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26) but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2'',3''-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase and the Ki [inhibition constant] values for the four 2'',3''-dideoxynucleoside triphosphates were determined for the 4 viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2'',3''-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to 3 DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 MW and defined an active center of the HSV DNA polymerase enzyme.