SeMNPV genotypic variants with increased replication efficiency in cultured Spodoptera exigua cells lack a gene with pro-apoptotic activity

Abstract

The Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate SeUS1 is a complex mixture of genotypes. SeUS1 viral DNA was used for the construction of bacmids by direct cloning in E. coli. A complete bacmid (SeBac10) and several variants were generated. Variant SeBac72 retained oral infectivity in larvae but had a much higher transfection efficiency in Se301 and SeUCR cells as compared to SeBac10. Moreover, SeBac10 serial passaging in cell culture generated large deletions in the region ORF15-41, whereas SeBac72 remained stable. We hypothesized that one or more gene(s) in the SeBac72 deleted region prevents successful virus replication in cell culture. Genome sequencing of SeBac72 revealed a 9.5 kb deletion spanning ORF16-28. Transient expression in Sf21 cells showed that one of these ORFs induced the formation of apoptotic bodies and upregulated effector caspase activity. These findingsmake this gene a candidate responsible for the observed lower transfection efficiency of SeBac10. Bacmids with different ORF16-28 knockouts were constructed to assess transfection efficiency and the induction of apoptosis in S. exigua cells. These results indicate that baculoviruses, which clearly rely on anti apoptotic genes for replication, may also express proteins with pro apoptotic activity in the in vivo viral life cycle.