DNA-Replication Complex from Cells Infected with Herpes Virus

Abstract

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.