Aphidicolin, a tetracyclic diterpenoid which inhibits the DNA polymerase-α activities of many eukaryotic cells, inhibited herpes simplex virus growth and DNA synthesis in infected cultures and the activity of the virus DNA polymerase . A wide range of stable aphidicolin sensitivities was represented amongst a collection of virus strains with no prior exposure to this drug, but viruses with polymerase mutations selected for resistance to phosphonoacetic acid (PAA) or to acycloguanosine typically showed increased sensitivity to aphidicolin. Of 16 unrelated PAA-resistant variants, 7 were hypersensitive to aphidicolin. A number of mutants with temperature-sensitive () lesions in the polymerase gene also showed increased aphidicolin sensitivity (e.g. HSV-1[mP17]H) or aphidicolin hypersensitivity (e.g. HSV-1[KOS]D9, C4). Resistance or hypersensitivity of virus growth and DNA synthesis were correlated with resistance or hypersensitivity of virus DNA polymerase reactions . Resistance phenotypes were closely linked to the polymerase gene during recombination with outside markers. Moreover, the selection of aphidicolin-resistant mutants from hypersensitive variants with independent PAA resistance or mutations in the polymerase gene could result in co-selection for PAA-sensitive and phenotypes. Confirmation that multiple independent mutations could determine aphidicolin hypersensitivity was obtained by studies of recombination between independent hypersensitive variants. Aphidicolin-resistant recombinant progeny were formed with recombination frequencies (0.4 to 2.6%) compatible with intragenic events. With parental hypersensitive variants which were products of limited PAA selection, or with the polymerase mutations, aphidicolin-resistant recombinants were PAA-sensitive and/or . The segregation of other markers (, plaque morphology) amongst recombinant progeny permitted the orientation of multiple determinants of PAA resistance and aphidicolin hypersensitivity with respect to other markers in the polymerase gene and in other genes. The nature of residues determined at any one of a constellation of separate sites within the polymerase locus can determine resistance or sensitivity to antiviral drugs and aphidicolin hypersensitivity associated with changes at the polymerase locus facilitates high resolution genetic analysis of this locus.


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