From earlier studies it is known that 9-cy-D-arabinofuranosyladenine ((Y-araA) and 9-cY-D-arabinofuranosyl-8-azaadenine (cu-ara-8-azaA) have in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in uiuo antiviral activities of these compounds and their loci of action with those of 9-j?-D-arabinofuranosyladenine (/3-araA). The latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas /?-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, cu-araA and a-ara-8-azaA were therapeutically ineffective. cu-AraATP at a concentration of-1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-cufrom mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerasej3 from mouse embryo cells was inhibited to a small extent by 10e4M cY-araATP. In contrast, all of these enzymes were inhibited by /3-araATP at a concentration of 10m5M (as shown in these or in earlier studies). The reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by LY-araATP (-10e3M), whereas /3-araATP produced 70-80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, a+ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. a-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of ['"Cl formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and ['"Cl formate. These results indicate that the cY-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of /3-araA.

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.

Preliminary studies of the biochemical basis for the antiviral activity of the pyrrolo[2,3-d]pyrimidine nucleoside ara-tubercidin were conducted. Herpes simplex virus DNA synthesis was 3-fold more sensitive to inhibition by ara-tubercidin than was cellular DNA synthesis. Partially purified herpes DNA polymerases were more sensitive to inhibition by ara-tubercidin 5'-triphosphate than were cellular polymerases alpha and beta. Inhibition of viral DNA polymerase was competitive with dATP and noncompetitive with dTTP. The results suggest that the viral DNA polymerase plays a significant role in the antiviral activity of ara-tubercidin.

We have developed a simplified assay for the enzyme N-acetyltransferase, based upon the loss of fluorescence after acetylation of the substrate p-aminobenzoic acid. This method is sufficiently sensitive to permit the quantitation of N-acetyltransferase activity in 10(5) human lymphocytes. Using this method, we have compared the level of N-acetyltransferase activity in lymphocytes from adult peripheral blood and from cord blood samples.