Treatment of cells with combinations of human interferon-alpha (IFN-alpha) and the nucleoside analog, acyclovir (ACV), leads to the synergistic inhibition of herpes simplex virus type 1 (HSV-1) replication. We have examined the effect of these agents on the replication of HSV-1 DNA and the synthesis of early viral enzymes to understand the mechanism(s) responsible for this synergistic activity. Combination treatment with 100 IU/ml IFN-alpha and 5 microM ACV led to HSV-1 DNA levels more than 8-fold lower than in cells treated with ACV alone, while IFN-alpha treatment alone had no detectable effect on viral DNA synthesis. Steady state levels of DNA polymerase were reduced approximately 50% by IFN-alpha and 25% by ACV, but combination treatment did not decrease enzyme levels to an extent greater than the sum of these effects. In contrast, the activity of another early viral enzyme, alkaline DNase, was reduced less than 20% by IFN-alpha alone or combination treatment and was unaffected by ACV treatment. No decrease in the level of mRNA encoding either enzyme was detected in IFN-alpha-treated cells although ACV treatment reduced polymerase mRNA levels. These studies suggest that the synergistic anti-HSV activities of IFN-alpha with ACV could be mediated, in part, through some post-transcriptional mechanism induced by IFN-alpha treatment, leading to the reduction in production of viral early enzymes, especially DNA polymerase.