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

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.

依赖感染的复制实验已被用于鉴定杆状病毒复制的众多潜在起源位点。然而，当质粒DNA与苜蓿银纹夜蛾多核衣壳核型多角体病毒（Autographa californica multinucleocapsid nucleopolyhedrovirus, AcMNPV）DNA共转染昆虫细胞时，其复制可不依赖于任何顺式作用的病毒序列[11]。转移质粒与杆状病毒DNA的共转染是生成重组病毒以及在瞬时表达实验中检测基因表达水平的常规实验手段。本研究对一系列载体质粒在共转染实验中的命运进行了考察。实验数据表明，这些质粒在共转染后可发生复制，且质粒DNA的复制并非源于获得了病毒的潜在起源序列。对共转染细胞中复制的质粒DNA构象进行分析后发现，其以高分子量多联体的形式存在。10%至25%的复制型质粒DNA会整合至病毒基因组的多个位点，并在连续传代后留存于子代病毒粒子中。对质粒-病毒DNA连接位点的序列分析显示，整合位点附近不存在同源或保守序列，表明整合过程涉及非同源重组。上述数据表明，尽管杆状病毒DNA复制可能采用滚环复制机制，但重组过程也可能参与其中。质粒整合可能会造成病毒基因组的大片段缺失，这提示杆状病毒的DNA复制过程易于产生缺陷型基因组。