Genetic Requirements for Homologous Recombination in Autographa californica Nucleopolyhedrovirus

Abstract

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.Studies on homologous recombination (HR) among baculovirus genomes in cell culture and in the wild are limited, curtailing our understanding of genetic diversity among baculovirus strains and virus evolution and our ability to design ecologically safe and efficient biological control agents. Recombination of virus genomes within insects results in an increase in virus genetic heterogeneity in wild populations (11-13, 41, 45, 60) and in cell culture (17,23,61,71). Integration of plasmid DNA by nonhomologous recombination into the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) DNA has also been observed in cell culture (69). In addition to shedding light on these processes, identification of the viral genes involved in HR may be useful for studying the function of genes in the host organism by specifically targeting genes in insects infected with baculoviruses or other viruses carrying baculovirus recombination-specific genes. For example, gene targeting mediated by baculoviruses in the silkworm Bombyx mori has been successful (72). Also, knowledge of the mechanism of recombination in the host organism may provide a tool to better develop recombination between baculovirus and plasmid DNA after lipofection of insect larvae. The latter has been suggested and investigated to generate recombinant viruses in the event an insect cell line i...

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The Highly Conserved Basic Domain I of Baculovirus IE1 Is Required for hr Enhancer DNA Binding and hr -Dependent Transactivation

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Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation

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