Differential activity of two non-hr origins during replication of the baculovirus Autographa californica nuclear polyhedrosis virus genome

The identification of potential baculovirus origins of replication (ori) ha s involved the generation and characterization of defective interfering par ticles that contain major genomic deletions Set retain their capability to replicate by testing the replication ability of transiently transfected pla smids carrying viral sequences in infected cells. So far, there has not bee n any evidence to demonstrate the actual utilization of these putative orig ins indrttographa californica multinueleocapsid nuclear polyhedrosis virus (AcMNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for the ori activity of the HindIII -K region and the ie-1 promoter sequence AcMNPV. We also provide evidence f or differential activity of the two ori in the context of the viral genome through the replication phase of viral infection. Comparison of the number of molecules representing the HindIII-K and ie-1 origins vis-g-vis the non- ori polH region in a size-selected nascent DNA preparation revealed that th e HindIII-K ori is utilized similar to 14 times more efficiently than the i e-1 region during the late phase of infection. HindIII-K also remains the m ore active ori through the early and middle replication phases. Our results provide in vivo evidence in support of the view that AcMNPV replication in volves multiple ori that are activated with vastly different efficiencies d uring the viral infection cycle.