Initiation of baculovirus DNA replication: early promoter regions can function as infection-dependent replicating sequences in a plasmid-based replication assay.

From the results of transient plasmid-based replication assays, it has been postulated that homologous regions (hrs) of Autographa californica nuclear polyhedrosis virus (AcMNPV) function as origins of viral DNA replication. However, these assays vary in specificity according to the methodology used and may not be dependent solely on the presence of hr sequences. To determine the role that hrs and other sequences might play in the replication process, a series of plasmids containing specific deletions of various hrs was generated and tested in a standardized replication assay. Deletion of the AcMNPV hr2 and hr5 sequences abolished the ability of plasmids to replicate in the standard infection-dependent replication assay, while deletion of hr1, hr3, and hr4a sequences decreased but did not eliminate plasmid replication in this assay. Plasmids carrying the complete ie-2 and pe38 genes, the ie-1 gene upstream region, or a variety of baculovirus genes including 11 early promoter regions were also able to replicate in virus-infected cells, suggesting that early viral promoter sequences could also function as putative origins of replication. These data suggest that the standard infection-dependent replication assay may identify a broad range of infection-dependent replicating sequences, only one or a few of which may represent genuine viral origins used by the virus in vivo. We propose a model suggesting that the selection of replication initiation sites may be imposed directly by chromatin structure and indirectly by primary sequence and that the process of viral DNA replication may be linked with viral transcription.