DNA Replication Promotes High-Frequency Homologous Recombination duringAutographa californicaMultiple Nuclear Polyhedrosis Virus Infection

Abstract

The relative ease with which foreign genes can be incorporated into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) indicates that a highly efficient recombinational process exists within infected cells. However, it is unclear whether this is due to marker transfer mediated by host cell enzymes or recombination events promoted by AcMNPV itself. To address the latter possibility, a pair of inverted repeat IS50 elements derived from the bacterial transposon Tn5 was inserted into the polyhedrin gene locus of the AcMNPV genome. Inversion of Tn5 sequences arising from recombination between its IS50 repeats could be readily detected in this virus, indicating that AcMNPV DNA undergoes high-frequency recombination during infection. To further characterize this process, a transient recombination assay was developed and used to identify the cis- and trans-acting requirements for Tn5 inversion in AcMNPV. A transfected Tn5-containing plasmid was found to undergo the same sequence inversion events seen in the viral genome, but only if it also contained a putative AcMNPV origin of replication (homologous region 2) in cis and was replicated by AcMNPV gene products supplied in trans. Taken together, these results indicated that recombination events which occur in infected cells were strictly dependent upon AcMNPV-mediated DNA replication. Direct support for this hypothesis was provided by the observation that the minimal set of AcMNPV genes that was essential for plasmid DNA replication also promoted recombination events leading to Tn5 inversion in the absence of any other viral function. Finally, using a panel of deletion mutants of the IS50 elements in Tn5, sequence inversion was shown to be the result of homologous rather than site-specific recombination, since it occurred independently of a discrete sequence within the transposon. These results demonstrate that the AcMNPV DNA replication machinery exhibits a strong propensity to promote homologous recombination events during infection and is likely to play a role in the high frequency of marker transfer observed in this virus.

Genetically variable nucleopolyhedroviruses isolated from spatially separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) in Orkney

“…have all been observed in baculovirus systems (Crozier and Riberio, 1992;Crozier et al, 1988;Martin and Weber, 1997). With greater amounts of viral DNA present in a population, these processes may contribute towards forming new genotypic variants.…”

Genetic Requirements for Homologous Recombination in Autographa californica Nucleopolyhedrovirus

“…We were interested in defining the specific roles of AcMNPV genes involved in viral DNA replication in HR since it had been reported previously that HR in insect cells was dependent on DNA replication by supplying AcMNPV DNA replication genes in trans and a viral origin of replication in cis (38). To this end, we constructed two control plasmids and four sets of plasmids, each consisting of two HR targets (Fig.…”

“…In cell culture, recombination between plasmids carrying a foreign gene and baculovirus DNA has been exploited to construct recombinant viruses overexpressing a foreign gene; however, the involvement of viral genes in this process had not been investigated until recently. The only study that has examined the requirement of specific baculovirus genes in HR concluded that the AcMNPV DNA replication genes ie-1, lef-1, lef-2, lef-3, p35, and dnapol were necessary for high-frequency HR in the presence of an origin of viral replication, whereas ie-2 and lef-7 had little effect as monitored by the inversion of sequences within a bacterial transposable element (38).…”

OneBac 2.0:Sf9 Cell Lines for Production of AAV5 Vectors with Enhanced Infectivity and Minimal Encapsidation of Foreign DNA

“…Baculovirus replication promotes high-frequency homologous recombinations assumedly exploiting the repeated hr elements as cis signals. 31 We therefore assume that comparable recombination events occur in baculovirus-infected OneBac cell lines (Fig. 4).…”