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9-beta-D-Arabinofuranosyladenine 5'-triphosphate (ara-ATP) is an inhibitor both of DNA polymerase-alpha and -beta from noninfected rabbit kidney cells and of the DNA-dependent DNA polymerase induced by herpes simplex virus Type 1 (strain IES). The studies were performed with partially purified enzymes, and each of the different polymerase preparations contained only one DNA-dependent DNA polymerase species. These enzymes were inhibited in a competitive manner. The HSV-induced DNA-dependent DNA polymerase was 39-fold more sensitive to ara-ATP than was cellular DNA polymerase-beta and 116-fold more sensitive than cellular DNA polymerase-alpha. The affinity of the HSV-induced enzyme for ara-ATP was only slightly influenced by the use of different template/initiators in the enzyme assays. In intact cell systems DNA synthesis was affected by 9-beta-D-arabinofuranosyladenine (ara-A) as indicated by the reduced incorporation of deoxythymidine. In herpesvirus-(strain Lennette)-infected cells, however, ara-A shows no influence on the incorporation on deoxythymidine into cellular DNA, but it substantially reduces the incorporation into viral DNA. Ara-A itself is incorporated into both cellular and herpesviral (strain Lennette, D-316 and IES) DNA during DNA synthesis.

The influence of 9-beta-D-arabinofuranosyladenine (beta araAdo) and of its anomer 9-alpha-D-arabinofuranosyladenine (alpha araAdo) was studied in non-infected cells and cells infected with herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2). alpha AraAdo is a strong inhibitor of proliferation of non-infected cells. Multiplication of HSV-1 and HSV-2 is not affected at all by alpha araAdo, while their growth is strongly inhibited by beta araAdo. alpha AraAdo exerts no effect on the incorporation of dThd into HSV DNA, but blocks the incorporation into host cell DNA. Its anomer, beta araAdo, affects the incorporation rate of both the viral DNA system and the host cell DNA system (the latter one to a lesser extent). alpha AraAMP is incorporated into newly synthesized cellular DNA but not into HSV DNA. Enzymic studies relevant that alpha araATP has no effect on the HSV DNA polymerase system but a high inhibitory potency in the host cell DNA polymerase alpha system. The anomeric form, beta araATP, is a sensitive inhibitor of HSV DNA polymerase while the cellular DNA polymerases alpha and beta are more refractory.

Experiments about the interaction of herpes viruses with BHK-cells during the first 6 h after infection concerning uptake and incorporation of dThd have been reported. During adsorption and penetration, the inhibition of uptake and of incorporation of [3H] dThd is sensitive to heat, but not to ultraviolet irradiation or cycloheximide. The eclipse is characterized by a strongly increased uptake of [3H] dThd and by inhibition of cell DNA synthesis. Both are sensitive to ultraviolet irradiation of the particles and cycloheximid treatment of the cells. It is concluded that the events during adsorption and penetration are dependent on the particles themselves, whereas the events during the eclipse depend on the activity of the viral genome. The implications of the findings are discussed.

Herpes simplex virus (HSV) DNA synthesis is initiated in an intact cell system by a 36-residue ribonucleotide stretch [W.E.G. Müller, R.K. Zahn, J. Arendes, and D. Falke (1979) Virology, 98, 200-210]. In the present study a nucleoplasmic fraction was isolated from rabbit kidney cells infected with HSV (type 1), which catalyzes DNA synthesis. By means of specific assays, containing single-stranded deoxyribopolymers, it was elucidated that the replication complex contains both an RNA-synthesizing and a DNA-synthesizing enzyme. These enzymes were characterized as host cell RNA polymerase II and HSV-induced DNA polymerase. The RNA polymerase II synthesizes an RNA initiator with an average chain length of 25 nucleotides, to which the newly synthesized DNA is covalently attached. The chemical nature of the RNA primer was proven by degradation experiments with endoribonuclease V. In the absence of any initiator in the reaction mixture the HSV-induced DNA polymerase is inactive. Kinetic experiments revealed that DNA synthesis in assays containing poly(dT), dNTPs and NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA NTPs starts after a lag phase of 10 min during which the RNA initiator is synthesized. The HSV DNA replication complex was further fractionated into HSV-induced DNA polymerase and the three cellular RNA polymerases. In the reconstructed system DNA synthesis, catalyzed by virus-induced DNA polymerase starts in a reaction assay, containing single-stranded DNA template, only in the presence of RNA polymerase II. The conclusion that the subnucleoplasmic complex, isolated from HSV-infected cells, is of biological significance for HSV DNA synthesis stems (a) from the the close correlations of the known biochemical events occurring during the initiation of HSV DNA synthesis in intact cells and in the nucleoplasmic system and (b) from control experiments with a subnucleoplasmic complex, isolated from uninfected cells, which is devoid of any potency to initiate DNA synthesis.