Characterization of a Baculovirus alkaline nuclease

All baculovirus genomes sequenced to date encode a homolog of an alkaline n uclease that has been characterized in the Herpesviridae, In this report we describe the characterization of the alkaline nuclease (AN) homolog of the Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) (op en reading frame 133). His-tagged AN constructs were expressed in recombina nt baculoviruses and affinity purified, and then their enzymatic activity w as characterized, AN was found to degrade linear DNA at alkaline pH, prefer red Mg2+ over Mn2+, had optimal activity at 35 degrees C, and did not appea r to have a salt requirement. To rule out contamination by the endogenous b aculovirus gene product or a cellular enzyme, point mutations were introduc ed into a highly conserved domain of the gene. These mutations were found t o markedly reduce or eliminate most of the activity of the affinity-purifie d enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis, AN was found to be expressed at low l evels by 12 h postinfection, with maximal expression at 24 h postinfection, Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.