Seven putative origins of DNA replication () were identified and located on the genome of multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV), when an improved infection-dependent replication assay was used. A threefold higher yield of amplified plasmid was achieved when an m.o.i. of 1 was used (instead of 25), and another twofold increase was obtained when the interval between transfection and infection was extended from 5 to 24 h. Six of the putative s were located in regions with homologous sequences. This suggests that all in AcMNPV are bifunctional, i.e. have both and enhancer activity for transcription. In addition to the six , the dIII-K fragment of AcMNPV was also identified to carry a putative , although this fragment does not contain an region. However, the individual role of these seven s during viral DNA replication, and whether they are all active simultaneously , is still unclear. The replication of an -containing plasmid starts at the same time (6 h post-infection) and proceeds at the same rate as viral DNA replication. A circular topology of -containing plasmids was a prerequisite for replication. Linear DNA, with an , did not replicate. Therefore, we suggest a theta structure or a rolling-circle as a model for baculovirus DNA replication.


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