Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus.
It is known that baculovirus infection promotes high-frequency recombination between its genomes and plasmid DNA during the construction of recombinant viruses for foreign gene expression. However, little is known about the viral genes necessary to promote homologous recombination (HR). We developed an assay to identify viral genes that are necessary to stimulate HR. In this assay, we used two plasmids containing extensive sequence homology that yielded a visible and quantifiable phenotype if HR occurred. The plasmids contained the green fluorescent protein gene (gfp) that was mutated at either the N or the C terminus and a viral origin of DNA replication. When the plasmids containing these mutant gfp genes were transfected into insect cells alone or together, few green fluorescent protein (GFP)-positive cells were observed, confirming that the host cell machinery alone was not able to promote high levels of HR. However, if viral DNA or viral genes involved in DNA replication were cotransfected into cells along with the mutant gfp-containing plasmids, a dramatic increase in GFP-positive cells was observed. The viral genes ie-1, ie-2, lef-7, and p35 were found to be important for efficient HR in the presence of all other DNA replication genes. However, ie-1 and ie-2 were sufficient to promote HR in the absence of other viral genes. Recombination substrates lacking a viral origin of replication had similar genetic requirements for recombination but were less dependent on ie-1. Interestingly, even though HR was stimulated by the presence of a viral origin of DNA replication, virally stimulated HR could proceed in the presence of the DNA synthesis inhibitor aphidicolin.
Crouch
Erin A
EA
Division of Biology, Molecular, Cellular, and Developmental Biology Program, Kansas State University, Manhattan, Kansas 66506, USA.
Passarelli
A Lorena
AL
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D002460:Cell Line; D004261:DNA Replication / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D005814:Genes, Viral; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000187:drug effects / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D018741:Replication Origin; D018411:Spodoptera; D014162:Transfection; D014773:Virology / Q000379:methods
D000998:Antiviral Agents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D016590:Aphidicolin