Inhibition of Herpes Simplex Virus Replication by Phosphonoacetic Acid

Abstract

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

Roscovitine, a Specific Inhibitor of Cellular Cyclin-Dependent Kinases, Inhibits Herpes Simplex Virus DNA Synthesis in the Presence of Viral Early Proteins

“…As a first test of this hypothesis, we asked whether Rosco can inhibit HSV replication after release from a PAA block. PAA is a well-characterized inhibitor of HSV DNA polymerase (3,29,36,42,53,61). In the presence of PAA, viral IE and E proteins are synthesized but the activity of the viral DNA Pol (an essential E protein) is directly inhibited by the drug.…”

(E)-5-(2-Bromovinyl)-2'-deoxyuridine: a potent and selective anti-herpes agent.

“…In recent years several compounds have been described which are all endowed with selective anti-herpes properties. These compounds include phosphonoacetic acid (3,4), phosphonoformic acid (5, 6), 9-f3-D-arabinofuranosyladenine (araA) (7,8), 5-iodo-5'-amino-2',5'-dideoxyuridine (AIDDU) (9, 10), 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) (11,12), 1-f3-D-arabinofuranosylthymine (araT) (13,14), 5-iodo-and 5-bromo-2'-deoxycytidine (15, 16), 5,6-dihydro-5-azathymidine (17,18), and erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) (19). Among the 5-substituted 2'-deoxyuridines, various compounds-namely, 5-methylamino-dUrd (20), 5-methoxymethyl-dUrd (21), 5-propyl-dUrd (22), and 5-propynyloxy-dUrd (23)-proved more inhibitory to herpes than to any other DNA (or RNA) virus.…”

A Herpes Simplex Virus Scaffold Peptide That Binds the Portal Vertex Inhibits Early Steps in Viral Replication

“…To further characterize the defect in viral replication, cells were infected and treated or left untreated with mutant or wild-type peptides at 30 min after infection in the presence of absence of phosphonoacetic acid (PAA), a potent inhibitor of the viral DNA synthesis (57). Cells were then fixed at 3 h, and DNA was dena- resembled that of cells infected with HSV-1(F) that were treated with PAA.…”