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

Adam L Vanarsdall1, Kazuhiro Okano, George F Rohrmann

  • 1Department of Microbiology, Nash Hall Room 220, Oregon State University, Corvallis, OR 97331-3804, USA.

Virology
|December 8, 2004
PubMed

Insights

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase is essential for viral DNA replication. A dnapol-null virus could not replicate DNA, confirming the viral polymerase

Area of Science:

  • Molecular Virology
  • Insect Virology
  • Baculovirus Research

Background:

  • Conflicting reports exist on the essentiality of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase for viral DNA replication.
  • Previous studies utilized transient replication assays with differing conclusions regarding the role of the viral DNA polymerase.

Purpose of the Study:

  • To resolve conflicting data regarding the AcMNPV DNA polymerase's role in viral DNA replication.
  • To determine if the viral DNA polymerase is strictly required during a productive infection cycle.

Main Methods:

  • Generation of a dnapol-null AcMNPV using bacmid technology.
  • Development of a sensitive real-time PCR assay with SYBR green chemistry to detect viral DNA replication.
  • Transfection of Spodoptera frugiperda (Sf-9) cells with wild-type, dnapol-null, and repaired viruses.

Main Results:

  • A bacmid lacking the dnapol open reading frame (ORF) failed to replicate its DNA in Sf-9 cells.
  • Reintroduction of the dnapol ORF into the polyhedrin (polh) locus restored viral DNA replication to levels comparable to the control virus.
  • The AcMNPV-encoded DNA polymerase is indispensable for viral DNA replication.

Conclusions:

  • The AcMNPV DNA polymerase is essential for viral DNA replication.
  • Host DNA polymerases cannot compensate for the absence of the viral DNA polymerase.
  • This study confirms the critical role of the viral DNA polymerase in the baculovirus life cycle.

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