Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene

Adam Vanarsdall, Kazuhiro Okano, George F. Rohrmann

Research output: Contribution to journalArticle

80 Citations (Scopus)

Abstract

In a previous study, the DNA polymerase gene (dnapol) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was identified as one of six genes required for plasmid replication in a transient replication assay (M. Kool, C. Ahrens, R.W. Goldbach, G.F. Rohrmann, J.M. Vlak, Identification of genes involved in DNA replication of the Autographa californica, Proc. Natl. Acad. Sci. U.S.A. 91, (1994) 11212-11216); however, another study based on a similar approach reported that the virally encoded polymerase was only stimulatory (A. Lu, L.K. Miller, The roles of 18 baculovirus late expression factor genes in transcription and DNA replication, J. Virol. 69, (1995) 975-982). To reconcile the conflicting data and determine if the AcMNPV DNA polymerase is required for viral DNA replication during the course of an infection, a dnapol-null virus was generated using bacmid technology. To detect viral DNA replication, a highly sensitive assay was designed based on real-time PCR and SYBR green chemistry. Our results indicate that a bacmid in which the dnapol ORF was deleted is unable to replicate its DNA when transfected into Spodoptera frugiperda (Sf-9) cells, although when the dnapol ORF was introduced into the polyhedrin (polh) locus, this repaired virus could propagate at levels similar to the control virus. These results confirm that the AcMNPV-encoded DNA polymerase is required for viral DNA replication and the host DNA polymerases cannot substitute for the viral enzyme in this process.

Original languageEnglish (US)
Pages (from-to)175-180
Number of pages6
JournalVirology
Volume331
Issue number1
DOIs
StatePublished - Jan 5 2005
Externally publishedYes

Fingerprint

Baculoviridae
DNA-Directed DNA Polymerase
DNA Replication
Nucleopolyhedrovirus
Viral DNA
Genes
Viruses
Open Reading Frames
Sf9 Cells
Real-Time Polymerase Chain Reaction
Plasmids
Technology
Gene Expression
DNA
Enzymes
Infection

Keywords

  • AcMNPV dnapol knockout bacmid
  • Baculovirus replication

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

Cite this

Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene. / Vanarsdall, Adam; Okano, Kazuhiro; Rohrmann, George F.

In: Virology, Vol. 331, No. 1, 05.01.2005, p. 175-180.

Research output: Contribution to journalArticle

Vanarsdall, Adam ; Okano, Kazuhiro ; Rohrmann, George F. / Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene. In: Virology. 2005 ; Vol. 331, No. 1. pp. 175-180.
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