Internucleotide incorporation of arabinosyladenine into herpes simplex virus and mammalian cell DNA

Abstract

Viral DNA was isolated from lysates of herpes simplex virus type 1-infected cells which had been incubated in the presence of 0.1, 1.0, or 3.2 μM [ 3H]ara-A. The DNA was purified by isopycnic centrifugation and was enzymatically digested with micrococcal nuclease and spleen phosphodiesterase. The resulting nucleotides and nucleosides were resolved by means of thin-layer chromatography. Examination of the distribution of radioactivity on the chromatograms revealed that 92–96% of the label was present as nucleotides demonstrating that [ 3H]ara-A was internally incorporated into viral DNA. Virtually identical results were obtained using DNA isolated from mature virions. Additional analysis of virion DNA using HindIII restriction endonuclease demonstrated that the incorporation of [ 3H]ara-A into the viral genome was random and did not occur at preferential sites. The amount of ara-A incorporated into viral DNA from infected cells was compared with the amount incorporated into cellular DNA from uninfected cells. Viral DNA isolated from cells maintained in 0.1, 1.0, or 3.2 μM [ 3H]ara-A, contained 2.3, 9.7, or 27.5 ara-AMP residues/10,000 dAMP residues, respectively, whereas uninfected cellular DNA. contained 4.1, 41.0, or 160 ara-AMP residues/10,000 dAMP residues, respectively. These data establish that (i) ara-A is not an absolute chain terminator of HSV-1 DNA synthesis, and (ii) the amount of ara-A incorporated into viral DNA is less than the amount incorporated into cellular DNA.