Expression- and genomic-level changes during passage of four baculoviruses derived from bacmids in permissive insect cell lines

The baculovirus-based bacmid expression vector system has been widely used for protein production in basic research and biotechnological laboratories. Since the first construction of the Autographa californica multiple nucleopolyhedrovirus bacmid (AcBacmid), three more bacmids have been created from Bombyx mori nucleopolyhedrovirus (BmBacmid), Spodoptera exigua nucleopolyhedrovirus (SeBacmid) and Helicoverpa armigera nucleopolyhedrovirus (HaBacmid). Each of these bacmid-derived viruses replicates efficiently in a range of specific and permissive cell types. Here, we investigated the relative stability of each virus derived from the bacmid during passage in permissive cell lines through assessment of their expression level and genome structure changes. Using two different reporters, the expression levels of the viruses from the AcBacmid-Sf9, AcBacmid-Tn5, BmBacmid-BmN and SeBacmid-SeE1 bacmid-cell systems were significantly reduced after five passages of the viruses, whereas the reductions were not detected in the AcBacmid-Sf21 and HaBacmid-HzAM1 systems. Pulse field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis of viral DNA isolated from passaged viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 systems showed no major genomic changes. In contrast, the genomes from passaged viruses in the AcBacmid-Tn5 and AcBacmid-Sf9 systems displayed reduced genome size and various mutations at individual loci, including genotypes missing one at least or more viral RNA polymerase subunits and fp25k. These genotypic changes were correlated with reduced protein expression. RFLP analysis of viral DNA from passaged viruses in the BmBacmid-BmN and SeBacmid-SeE1 systems exhibited changes in genome size, including excision of particular EcoRI fragments containing the mini-F replicon. Collectively, our data suggest that the viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 bacmid-cell systems are better for large-scale protein expression in continuous culture. Further study is needed to investigate the mechanism(s) behind the protein expression reduction in these bacmid-derived virus/cell systems.