DNA-REPLICATION PROMOTES HIGH-FREQUENCY HOMOLOGOUS RECOMBINATION DURING AUTOGRAPHA-CALIFORNICA MULTIPLE NUCLEAR POLYHEDROSIS-VIRUS INFECTION

The relative ease with which foreign genes can be incorporated into th e genome of the baculovirus Autographa californica nuclear polyhedrosi s Virus (AcMNPV) indicates that a highly efficient recombinational pro cess exists within infected cells. However, it is unclear whether this is due to marker transfer mediated by host cell enzymes or recombinat ion events promoted by AcMNPV itself. To address the latter possibilit y, a pair of inverted repeat IS50 elements derived from the bacterial transposon Tn5 was inserted into the polyhedrin gene locus of the AcMN PV genome. Inversion of Tn5 sequences arising from recombination betwe en its IS50 repeats could he readily detected in this virus, indicatin g that AcMNPV DNA undergoes high-frequency recombination during infect ion. To further characterize this process, a transient recombination a ssay was developed and used to identify the cis- and trans-acting requ irements for Tn5 inversion in AcMNPV. A transfected Tn5-containing pla smid was found to undergo the same sequence inversion events seen in t he viral genome, but only if it also contained a putative AcMNPV origi n of replication (homologous region 2) in cis and was replicated by Ac MNPV gene products supplied in trans. Taken together, these results in dicated that recombination events which occur in infected cells were s trictly 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 als o promoted recombination events leading to Tn5 inversion in the absenc e of any other viral function. Finally, using a panel of deletion muta nts of the IS50 elements in Tn5, sequence inversion was shown to be th e 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 event s during infection and is likely to play a role in the high frequency of marker transfer observed in this virus. (C) 1997 Academic Press.