A New DNA-exonuclease in Cells Infected with Herpes Virus: Partial Purification and Properties of the Enzyme

Abstract

SUMMARYInfection of BHK 2I (C 13) or HEp-2 cells with herpes virus was followed by a marked increase in the activity of alkaline DNase which was prevented by actinomycin D and puromycin. Activation of a latent enzyme was not responsible for the increase. The properties of the DNase appearing after virus infection in both cell types were the same, but they differed from those of the enzyme from uninfected cells in specificity towards the secondary structure of the DNA substrate, heat-stability, requirement for thiol groups, inhibition by K + and Na + ions and response to various concentrations of Mg ~+ and Mn ~+. The activities of acid DNase and alkaline phosphomonoesterase were not significantly altered after herpes infection. Further, the activity of alkaline RNase was not altered by infection, and this implies that the induced DNase was specific for DNA. The new DNase could be separated from the DNase present in uninfected cells and from alkaline phosphomonoesterase by chromatography on columns of DEAE-cellulose; its enzymic properties were the same as those observed with soluble extracts of cells infected with herpes virus.

The human cytomegalovirus UL98 gene encodes the conserved herpesvirus alkaline nuclease.

“…The alkaline nuclease was first described (Morrison & Keir, 1968), Author for correspondence : Daniel Tenney.…”

“…Enzymatic activity has not previously been demonstrated for a beta-herpesvirus alkaline nuclease, although the purified enzymes from the alpha-herpesviruses HSV-1 and HSV-2, and the gamma-herpesvirus Epstein-Barr virus (EBV) have been characterized in vitro (Morrison & Keir, 1968 ;Hoffman & Cheng, 1978 ;Bayliss et al, 1989 ;Knopf & Weishart, 1990 ;Stolzenberg & Ooka, 1990).…”

DNA sequence of the region in the genome of herpes simplex virus type 1 containing the exonuclease gene and neighbouring genes

“…The HSV proteins which participate in DNA replication include enzymes involved in the metabolism of DNA precursors (for instance, thymidine kinase and ribonucleotide reductase (4,5)), and also species more directly concerned with DNA synthesis. The latter include a DNA polymerase, an exonuclease and a polypeptide designated operationally as the "major DNA binding protein" (6,7,8). As part of a large scale sequence analysis of HSV-1 DNA, we recently reported the sequences of the genes for DNA polymerase and the major DNA binding protein (9).…”

“…The exonuclease was first detected as an increased alkaline nuclease activity present in HSV infected cells (10) and was subsequently purified and shown to be a single polypeptide chain (7,11,12,13), possessing both 5' and 3' exonuclease activity and also an endonuclease activity (11,14,15). Analysis of temperature sensitive mutants of the HSV-2 exonuclease gene has shown that the enzyme is necessary for efficient replication of virus DNA, although its precise role has not been defined (16,17,18).…”

Aspects of Thymidine Metabolism and Function in Cultured Mammalian Cells Infected with Herpes Simplex Virus Type 1

“…These include a new virus-coded DNA-dependent DNA polymerase, first observed by Keir & Gold (1963), a deoxypyrimidine kinase, with both thymidine and deoxycytidine kinase activities (Jamieson et al, 1974), an alkaline deoxyribonuclease 0022-1317/82/0000-4889 $02.00 © 1982 SGM H. N. BAYBUTT, B. A. MURRAY AND C. K. PEARSON (Morrison & Keir, 1968), deoxycytidine deaminase (Chan, 1977), CDP-reductase (Cohen, 1972) and possibly dCMP deaminase (Rolton & Keir, 1974), although other workers have not been able to demonstrate induction of this enzyme in herpesvirus-infected cells (Langelier et al, 1978;Huszar & Bacchetti, 1981). Concomitant with the induction of these enzyme activities, in particular the deoxypyrimidine kinase, exogenous thymidine is taken up into the cell and is rapidly phosphorylated before being incorporated into DNA (Jamieson & Bjursell, 1976a: Bittlingmaier et al, 1977.…”