DNase Induced After Infection of KB Cells by Herpes Simplex Virus Type 1 or Type 2 II. Characterization of an Associated Endonuclease Activity

Abstract

Purified preparations of the “exonuclease” specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg 2+ or Mn 2+ is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca 2+ , whereas no activity occurs in the presence of Zn 2+ . In the presence of Mg 2+ , Ca 2+ and Zn 2+ are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn 2+ greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg 2+ . The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg 2+ than Mn 2+ presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.