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Journal of Virology, October 2004, p. 10650-10656, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10650-10656.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of a Baculovirus Lacking the Alkaline Nuclease Gene

Kazuhiro Okano, Adam L. Vanarsdall, and George F. Rohrmann*

Department of Microbiology, Oregon State University, Corvallis, Oregon

Received 3 March 2004/ Accepted 7 May 2004

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5'->3' exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the {lambda} Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a bacmid containing the AcMNPV genome in Escherichia coli. The AcMNPV an knockout bacmid (vAcAN-KO/GUS) was unable to propagate in Sf9 cells, although an an-rescued bacmid (vAcAN-KO/GUS-Res) propagated normally. In addition, the mutant did not appear to produce budded virions. These data indicated that an is an essential baculovirus gene. Slot blot and DpnI assays of DNA replication in Sf9 cells transfected with vAcAN-KO/GUS, vAcAN-KO/GUS-Res, and a wild-type bacmid showed that the vAcAN-KO/GUS bacmid was able to replicate to levels similar to those seen with the vAcAN-KO/GUS-Res and wild-type bacmids at early stages posttransfection. However, at later time points DNA did not accumulate to the levels seen with the repaired or wild-type bacmids. Northern analysis of Sf9 cells transfected with bacmid vAcAN-KO/GUS showed that transcription of late and very late genes was lower at later times posttransfection relative to the results seen with wild-type and vAcAN-KO/GUS-Res bacmids. These data suggest that the an gene might be involved in the maturation of viral DNA or packaging of the DNA into virions.

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

Journal of Virology, October 2004, p. 10650-10656, Vol. 78, No. 19
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.19.10650-10656.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Knebel-Morsdorf, D., Quadt, I., Li, Y., Montier, L., Guarino, L. A. (2006). Expression of Baculovirus Late and Very Late Genes Depends on LEF-4, a Component of the Viral RNA Polymerase Whose Guanyltransferase Function Is Essential.. J. Virol. 80: 4168-4173 [Abstract] [Full Text]  


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