Sequential deletion of AcMNPV homologous regions leads to reductions in budded virus production and late protein expression

Homologous regions (hrs) have been predicted to act as origins of baculovirus DNA replication. Hrs have also been shown to function as enhancers of virus transcription. Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) carries eight hrs. In order to assess the role of hrs in virus replication in vivo, we applied a two-step RED recombination system for site-specific mutagenesis to sequentially delete each hr from a bacmid copy of AcMNPV. We then characterized the ability of the bacmids carrying different numbers of hrs or no hr to produce polyhedra and budded virus in transfected cells. We also investigated the ability of virus supernatants from transfected cells to produce budded virus and polyhedra when used to infect cells. We also characterized the expression of specific early and late virus proteins in transfected cells. The results demonstrated that removal of five hrs had little or no effect on virus infection but deleting all eight hrs compromised budded virus production and delayed early and late gene expression but did not completely eliminate assembly of infectious virus. We conclude that multiple hrs ensure an effective virus infection cycle with production of high titers of budded virus and polyhedra.