Replication of herpes simplex virus in WI-38 cells was inhibited by phosphonoacetic acid, as measured by decreased virus cytopathogenic effect and incorporation of radiolabeled thymidine in virus-infected cells. The drug appeared to have no effect on adsorption, penetration, or release of the virus nor on the synthesis of ribonucleic acid or protein. It appeared to inhibit virus deoxyribonucleic acid synthesis.Herpes simplex virus (HSV) is a frequent infection in man with degrees of severity from mild to severe discomfort (cutaneous lesions) to severe infection damaging the eye sight (herpes keratitis) to severe and life-threatening diseases (herpes encephalitis). It has been suggested recently that herpes-viruses may be directly or indirectly associated with cancer in several animals: frog renal adenocarcinoma (4), malignant lymphomas in chickens (Marek's disease) (11), rabbits (6), and monkeys (8). Herpes-type viruses have been indirectly associated with Burkitt's lymphoma, nasopharyngeal carcinoma (5), and cervical carcinoma (1) in humans. 5-Iodo-2-deoxyuridine and cytosine arabinoside are two well-known nucleoside analogs and are inhibitors of herpervirus replication. Both compounds are quite toxic (2, 12), and 5-iodo-2-deoxyuridine has been shown to be carcinogenic and teratogenic (10).In earlier studies by Shipkowitz et al. (13), phosphonoacetic acid (PAA) was shown to be active against herpes dermatitis in mice and herpes keratitis in rabbits. We report here the efficacy of PAA against HSV in tissue culture, its effect on WI-38 cells, and studies related to the mechanism of action. Infected cells. Virus stock solutions were diluted in MM and were added to confluent WI-38 cells. The virus was allowed to adsorb for 1 h at 37 C. The medium and unadsorbed virus were removed by decanting and the cells were washed twice with MM. Cell cultures were then incubated at 37 C in MM.Drug tests. PAA was prepared from sodium phosphite and sodium chloroacetic acid as described by Nylen (9). Stock solutions were prepared in MM at a concentration of 10 mg/ml. Portions were then added aseptically to cell cultures to give the desired final concentrations of PAA.Ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein synthesis. Cultures (1 ml) of WI-38 cells were grown and infected in tubes. At various intervals during the incubation, the cultures were pulse-labeled for 30 min with [14CIthymidine (0.5 MCi), [3Hjuridine (2.5 1Ci), or "4C-labeled amino acid mixtures (0.2 1Ci). PAA was added at the time of HSV adsorption and was continued in the culture medium thereafter. After each 30-min labeling with radioactive materials, triplicate tubes were removed, and then the medium was decanted. The cell layers were washed twice with cold MM. Then, 4 ml of phosphate-buffered saline were added to each tube, and the cells were scraped from the tube wall. Trichloroacetic acid was added to a final concentration of 5%. The mixture was frozen, thawed once, and

Phosphonoacetate is a highly specific inhibitor of herpes simplex virus-induced DNA polymerase. Sensitivity of herpesvirus type 1 or type 2 induced DNA polymerase to the drug was similar. However, DNA polymerases from other sources such as the host cells (Wi-38), Micrococcus luteus, and hepatitis B virus were highly resistant. In addition, Escherichia coli RNA polymerase and reverse transcriptase of Rous sarcoma virus were also insensitive to the drug. Enzyme kinetic studies showed that inhibition was noncompetitive with respect to deoxyribonucleotide triphosphates. The Ki value was about 0.45 muM. The apparent Km values for dTTP, dATP, dCTP, and dGTP were 0.71, 0.75, 0.42, and 0.39 muM, respectively. The base composition of template has no profound effect on the extent of inhibition. The drug caused uncompetititve inhibition with respect to template which indicated that phosphonoacetate did not bind directly to template DNA. Results are presented which suggest that phosphonoacetate did not affect the formation of the enzyme-DNA complex but probably inhibited the elongation step of DNA polymerase reaction.

Infection of Wi-38 cells with herpes simplex virus induced an elevated DNA polymerase activity which had many biochemical properties different from normal cell DNA polymerase. Phosphonoacetic acid specifically inhibited the virus-induced DNA polymerase as compared to the normal WI-38 cell DNA polymerase. The compound did not appear to inhibit enzyme activity by interacting with the DNA primer.

adenosine; GMPPCP, 5'-guanylylmethylenediphosphonate; 1 optical density unit is the amount of material which in 1.0 ml would yield a value of 1.0 for the optical density measured at 260 µ in a cuvet with a path length of 1.0cm.

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