Genetic mechanisms of resistance to acyclovir in herpes simplex virus

Abstract

Resistance to acyclovir in herpes simplex virus (HSV) is mediated by two genetic loci on the herpes virus genome: viral thymldine kinase and DNA polymerase. Passage of HSV in the presence of 10 μ M or 2 μ M acyclovir readily coselects for virus deficient in the expression of viral thymidine kinase and resistance to acyclovir. Other workers have shown that passage of HSV in serum-starved cells in the presence of acyclovir selects for a mutant virus possessing a thymidine kinase with an altered substrate specificity and inability to phosphorylate acyclovir. Mutants with temperature-sensitive lesions in the viral DNA polymerase loci of HSV also exhibit resistance to antiviral drug phosphonoacetic add (PAA) and acyclovir. The mutations conferring resistance to PAA and acyclovir are closely linked within the DNA polymerase locus. The map limits containing mutations thai confer resistance to PAA and acyclovir are found within a 1.3 kilobase pair (kbp) region of DNA sequences in HSV-2 (map unit 40.2 to 41.0) and within a 2.6 kbp region of DNA sequence in HSV-1 (map units 40.2 to 41.8). The DNA polymerase loci of HSV also contains a region of DNA sequences to the left of the region defining the resistance mutation which determines the type specific sensitivity of HSV-1 and HSV-2 to acyclovir. Two HSV-1/HSV-2 recombinants were selected for further analysis of resistance. Viral DNA polymerase enzymes were purified from the R6-26 and R6-34 recombinants; the resistant recombinant R6-34 was shown to process an altered viral DNA polymerase. This altered viral DNA polymerase exhibited resistance to three inhibitors of DNA polymerase activity, PAA, acyctovir- TP, and 2′ 3′ dideoxynucleotide triphosphate (didNTPs). The polymerase from R6-34 and the resistant parent possess a much higher K, for the PAA and the didNTP than do the sensitive strains. A 1.3 kbp region of HSV-1 DNA within the DNA polymerase locus contains resistance mutations to three DNA polymerase inhibitors, codes for an amino acid sequence of 42,000 molecular weight, and defines an active center of the HSV DNA polymerase enzymes.