Herpes simplex Virus Type 1 DNase: Functional Analysis of the Enzyme Expressed by Recombinant Baculovirus

Abstract

Herpes simplex virus type 1 DNase (HSV-1 DNase) was expressed in insect cells by recombinant baculovirus (NPVUL12) and purified by a combination of anionic exchanger chromatography and gel filtration. Two polypeptides of 85 and 75 kD, whose ratio varied during purification, were induced 24 h after infection. The 75-kD protein was isolated and shown to possess catalytic activity. Gel filtration analysis indicated that the active form of the enzyme at an ionic strength of I = 0.3 is a dimeric protein with an apparent molecular weight of 130,000. The recombinant enzyme exhibited the overall characteristics of the native enzyme such as 5′-3′ exonuclease and endonuclease activities with a preferred degradation of DNA. In the absence of extraneously added Mg²⁺, the enzyme was capable of removing mononucleotides from 5′-end-labeled DNA, but not from RNA and 3′-end-labeled DNA. The peculiar mechanism of double-strand DNA degradation suggests a specific role of HSV-1 DNase in DNA recombination processes during viral replication.