High-Frequency Homologous Recombination between Baculoviruses Involves DNA Replicationt

Shizuo George Kamita, Susumu Maeda, Bruce D. Hammock

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4% when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1. 4 kb, the mean frequency of recombination was 2.7%. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was ≲1.0%. AcMNPV DNA replication occurred normally after the coinfection of Sf-21 cells. BmNPV DNA replication, however, was not detected, indicating that normal DNA replication by both viruses is required for high-frequency recombination.

Original languageEnglish (US)
Pages (from-to)13053-13061
Number of pages9
JournalJournal of Virology
Volume77
Issue number24
DOIs
StatePublished - Dec 2003

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Bombyx mori nucleopolyhedrovirus
Nucleopolyhedrovirus
Bombyx
Baculoviridae
Homologous Recombination
homologous recombination
Genetic Recombination
Autographa californica multiple nucleopolyhedrovirus
DNA
Lac Operon
Heliothis virescens
DNA replication
DNA Replication
cells
Larva
genetic markers
Coinfection
mixed infection
larvae
Autographa californica

ASJC Scopus subject areas

  • Immunology

Cite this

High-Frequency Homologous Recombination between Baculoviruses Involves DNA Replicationt. / Kamita, Shizuo George; Maeda, Susumu; Hammock, Bruce D.

In: Journal of Virology, Vol. 77, No. 24, 12.2003, p. 13053-13061.

Research output: Contribution to journalArticle

Kamita, Shizuo George ; Maeda, Susumu ; Hammock, Bruce D. / High-Frequency Homologous Recombination between Baculoviruses Involves DNA Replicationt. In: Journal of Virology. 2003 ; Vol. 77, No. 24. pp. 13053-13061.
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abstract = "We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4{\%} when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1. 4 kb, the mean frequency of recombination was 2.7{\%}. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was ≲1.0{\%}. AcMNPV DNA replication occurred normally after the coinfection of Sf-21 cells. BmNPV DNA replication, however, was not detected, indicating that normal DNA replication by both viruses is required for high-frequency recombination.",
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