Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication

Abstract

We previously reported on the generation of approximately 50-kb size defective genomes (DGs) which appeared to retain less than 2.2% of the standard Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA between 85.0 and 87.2 MU while the rest of the virus DNA had been largely deleted (Lee and Krell, J. Virol., 66:4339-4347, 1992). To investigate these presumably repeated sequences further, we cloned and analyzed the most abundant hypermolar 1.80-kb Xhol DNA fragment as well as a minor but also supermolar 1.74-kb Xhol fragment of the DGs. These two DNA segments collectively covered 2371-bp of the standard AcMNPV DNA with a 1174-bp overlap around the Xhol site at 85.9 MU. Analysis of DGs by two-dimensional gel electrophoresis indicated that the 1.80- and 1.74-kb Xhol fragments (and most other novel Xhol fragments of the DG) were organized as tandem repeats in the DGs. We identified, in the DG population, small supercoiled DNA molecules approximately 1.0- to 8.2-kb (and possibly up to around 50 kb, the size of the major defective DNA species) in size and which contained the same DNA sequence as that of the major 1.80-kb repeat in the DGs. Furthermore, these cloned repeat sequences, represented by pLK1.80 and pLK1.74 showed AcMNPV infection-dependent autonomous replication, suggesting that an origin of DNA replication might reside within the HindIII to EcoRI segment (85.1 to 86.6 MU) of the HindIII-K fragment.