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J Virol. 1979 November; 32(2): 449-457

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

Peter J. Hoffmann and Yung-Chi Cheng

¹ Department of Experimental Therapeutics and Grace Cancer Drug Center, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263

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 Mg²⁺ or Mn²⁺ 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 Ca²⁺, whereas no activity occurs in the presence of Zn²⁺. In the presence of Mg²⁺, Ca²⁺ and Zn²⁺ are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn²⁺ greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg²⁺. 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 Mg²⁺ than Mn²⁺ 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.

Present address: Department of Medical Microbiology, The Ohio State University, Columbus, OH 43210.

Present address: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27514.

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