Characterization of an aphidicolin-resistant mutant of herpes simplex virus type 2 which induces an altered viral DNA polymerase.
The replication of wild-type herpes simplex virus type 2 (HSV-2) was very sensitive to aphidicolin, a specific inhibitor of eukaryotic alpha-type DNA polymerases; viral DNA synthesis was strongly inhibited by 1 microgram/ml of aphidicolin, but the synthesis of early viral polypeptides was not affected. Using aphidicolin as the selective agent, aphidicolin-resistant ( Aphr ) viruses were isolated from HSV-2 strain 186. All of these plaque isolates induced altered viral DNA polymerases which were more resistant to aphidicolin than wild-type polymerase. These results clearly indicate that viral DNA polymerase is a target of aphidicolin in vivo and suggest that host cell DNA polymerase alpha may be not involved in the replication of HSV-2. Partially purified mutant polymerase exhibited a 7.5-fold lower apparent Km for dCTP and a 3-fold lower apparent Km for dTTP than similarly purified wild-type enzyme. The apparent Ki value for aphidicolin of the mutant polymerase was 6.5-fold higher than that of the wild-type enzyme. Moreover, all Aphr viruses isolated were also resistant to thymine-1-beta-D-arabinofuranoside (ara-T). While, they were as sensitive as wild-type virus to cytosine-1-beta-D-arabinofuranoside (ara-C), adenine-9-beta-D-arabinofuranoside (ara-A), and acycloguanosine (acyclo-G). Interestingly these Aphr isolates were more sensitive to phosphonoacetic acid (PAA) than the wild-type. In contrast, PAA-resistant ( PAAr ) viruses of HSV-2 were more sensitive to aphidicolin and were more resistant to all of four nucleoside analogs than the parental wild-type virus. These results suggest that the aphidicolin-binding site of HSV DNA polymerase may be very close to the binding sites for dCTP and dTTP and it functionally correlates with that for pyrophosphate group.
D000998:Antiviral Agents / Q000633:toxicity*; D016590:Aphidicolin; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis*; D004224:Diterpenes / Q000633:toxicity*; D004352:Drug Resistance, Microbial; D004622:Embryo, Mammalian; D004790:Enzyme Induction; D005260:Female; D005347:Fibroblasts; D006801:Humans; D009154:Mutation; D011247:Pregnancy; D018139:Simplexvirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*
D000998:Antiviral Agents; D004279:DNA, Viral; D004224:Diterpenes; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase