title: Selection and characterization of Autographa californica multiple nucleopolyhedrovirus DNA polymerase mutations.
abstract: Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA polymerase (DNApol) is essential for viral DNA replication. AcMNPV mutants resistant to aphidicolin, a selective inhibitor of viral DNA replication, and abacavir, an efficacious nucleoside analogue with inhibitory activity against reverse transcriptase, were selected by the serial passage of the parental AcMNPV in the presence of increasing concentrations of aphidicolin or abacavir. These drug-resistant mutants had either a single (C543R) (aphidicolin) or a double (C543R and S611T) (abacavir) point mutation within conserved regions II and III. To confirm the role of these point mutations in AcMNPV DNA polymerase, a dnapol knockout virus was first generated, and several repair viruses were constructed by transposing the dnapol wild-type gene or ones containing a single or double point mutation into the polyhedrin locus of the dnapol knockout bacmid. The single C543R or double C543R/S611T mutation showed increased resistance to both aphidicolin and abacavir and, even in the absence of drug, decreased levels of virus and viral DNA replication compared to the wild-type repair virus. Surprisingly, the dnapol mutant repair viruses led to the generation of occlusion-derived viruses with mostly single and only a few multiple nucleocapsids in the ring zone and within polyhedra. Thus, these point mutations in AcMNPV DNA polymerase increased drug resistance, slightly compromised virus and viral DNA replication, and influenced the viral morphogenesis of occlusion-derived virus.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada.
authors.forename: Guozhong
authors.initials: G
authors.lastname: Feng
authors.forename: David K
authors.initials: DK
authors.lastname: Thumbi
authors.forename: Jondavid
authors.initials: J
authors.lastname: de Jong
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Hodgson
authors.forename: Basil M
authors.initials: BM
authors.lastname: Arif
authors.forename: Daniel
authors.initials: D
authors.lastname: Doucet
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Krell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D017354:Point Mutation; D060888:Real-Time Polymerase Chain Reaction; D012641:Selection, Genetic; D017386:Sequence Homology, Amino Acid; D061987:Sf9 Cells; D014779:Virus Replication
chemical_list: D004259:DNA-Directed DNA Polymerase