Characterization of a baculovirus lacking the alkaline nuclease gene

Journal of Virology
Kazuhiro OkanoGeorge F Rohrmann

Abstract

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-...Continue Reading

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Citations

Jun 23, 2010·Current Microbiology·Guohui LiXunzhang Wang
Aug 5, 2015·Journal of Virological Methods·Yanlong PeiÉva Nagy
Jul 1, 2011·International Journal of Evolutionary Biology·Solange Ana Belen MielePablo Daniel Ghiringhelli
May 9, 2008·Virus Research·Zhang-Nv YangChuan-Xi Zhang
Dec 21, 2005·Virology·Kazuhiro OkanoGeorge F Rohrmann
Jul 21, 2004·Virology·Adam L VanarsdallG F Rohrmann

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