High-frequency homologous recombination between baculoviruses involves DNA replication

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.

FIG. 1.

Linear maps of the circular genomes of viruses A to L. Each map initiates from the A (nucleotide 1) of the initiation codon (ATG) of the mutated (○) or wild-type (•) polyhedrin gene (polh). The open arrowhead indicates the relative location of the lacZ marker cassette of each virus with respect to polh. The numbers below the linear map indicate the location of the point mutation within polh or insertion coordinates of the lacZ marker cassette. The numbers in parentheses indicate the relative distances (in kilobases) between polh and the lacZ marker cassette of viruses B to L. The genotype of virus A is polh⁻ lacZ⁻. The genotype of viruses B to L is polh⁺ lacZ⁺. The ORF column indicates the BmNPV ORF (see reference for a complete list of BmNPV ORFs) that was inactivated by insertion of the lacZ marker cassette. If the ORF has been previously named, the name is given within the parentheses.

FIG. 2.

Linear maps of the progeny viruses that are released after coinfection of BmN cells with viruses A and G (A) or Sf-21 cells or larvae H. virescens with AcMNPV and virus G (B). Each map initiates from the mutated (○) or wild-type (•) polyhedrin gene. The open or filled arrows represent the lacZ marker cassette or wild-type ORF 74 locus, respectively.

FIG. 3.

Dot blot hybridization of total cell DNAs isolated from 10⁴ Sf-21 or BmN cells at 2, 12, 15, 18, or 24 h p.i. with AcMNPV and virus G (Ac+G), AcMNPV (Ac), virus G (G), viruses A and G (A+G), or virus A (A). The DNAs were fixed to a nylon membrane and hybridized with labeled AcMNPV DNA (NPV probe) or virus G-specific probe (lacZ probe). The standards consist of 0, 5, 20, 100, and 500 ng of AcMNPV DNA.