Characterization of an aphidicolin-resistant mutant of herpes simplex virus type 2 which induces an altered viral DNA polymerase

Abstract

The replication of wild-type herpes simplex virus type 2 (HSV-2) was very sensitive to aphidicolin, a specific inhibitor of eukaryotic α-type DNA polymerases; viral DNA synthesis was strongly inhibited by 1 μg/ml of aphidicolin, but the synthesis of early viral polypeptides was not affected. Using aphidicolin as the selective agent, aphidicolin-resistant (Aph r) 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 α may be not involved in the replication of HSV-2. Partially purified mutant polymerase exhibited a 7.5-fold lower apparent K m for dCTP and a 3-fold lower apparent K m for dTTP than similarly purified wild-type enzyme. The apparent K i value for aphidicolin of the mutant polymerase was 6.5-fold higher than that of the wild-type enzyme. Moreover, all Aph r viruses isolated were also resistant to thymine-l-β- d-arabinofuranoside (ara-T). While, they were as sensitive as wild-type virus to cytosine-1-β- d-arabinofuranoside (ara-C), adenine-9-β- d-arabinofuranoside (ara-A), and acycloguanosine (acyclo-G). Interestingly these Aph r isolates were more sensitive to phosphonoacetic acid (PAA) than the wild-type. In contrast, PAA-resistant (PAA r) 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.