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Journal of Virology, July 2000, p. 6401-6407, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of a Baculovirus Alkaline Nucleasedagger

Lulin Li and George F. Rohrmann*

Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804

Received 10 February 2000/Accepted 17 April 2000

All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease 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) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg2+ over Mn2+, had optimal activity at 35°C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified 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 levels 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.

* Corresponding author. Mailing address: Nash Hall 220, Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804. Phone: (541) 737-1793. Fax: (541) 737-0496. E-mail: rohrmann{at}bcc.orst.edu.

dagger Technical Report no. 11656 from the Oregon State University Agricultural Experiment Station.

Journal of Virology, July 2000, p. 6401-6407, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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