Replication, Integration, and Packaging of Plasmid DNA following Cotransfection with Baculovirus Viral DNA

Abstract

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.

No single homologous repeat region is essential for DNA replication of the baculovirus Autographa californica multiple nucleopolyhedrovirus

“…Therefore, each individual hr region in the viral DNA had been replaced by the lacZ gene. However, we have previously shown that plasmid DNA can become incorporated into BVs and can be continuously present in passaged virus stocks (Wu et al, 1999). We confirmed that the signals obtained from these PCRs were amplified from an integrated copy of the lacZ gene inserted into the corresponding region of the viral genome and not from a copy of the original plasmid co-packaged into virions and present as a contaminant in the infected cells with another series of primers designed from viral sequences lying outside the region present in the original plasmid transfer vectors (Supplementary Table S1, available in JGV Online).…”

Oligomerization of Baculovirus LEF-11 Is Involved in Viral DNA Replication

“…Baculoviruses are a highly diverse group of viruses with large, circular, double-stranded DNA genomes that exclusively infect invertebrates [ 1 , 2 ]. Specific genes have been identified that are required for viral DNA replication, and major advances have been made in understanding the functions of most of these genes in Alphabaculovirus ; however, the functional roles of DNA replication factors have not been fully elucidated [ 3 – 8 ].…”

Identification and Functional Analysis of BmNPV-Interacting Proteins From Bombyx mori (Lepidoptera) Larval Midgut Based on Subcellular Protein Levels

“…The microsome contains the endoplasmic reticulum, which is the main site for protein modification and processing, and serves as the processing station for intracellular proteins (Mandi et al, 2006). A large number of host proteins that are related to multiple biological processes, such as energy metabolism and apoptosis, are involved in the process of virus infection (Wu et al, 1999), which may explain the reason that the virus-binding proteins were identified in the mitochondria and microsome of the silkworm midgut and not in the cytosolic fraction.…”