Specific nucleotide substitutions in the burst sequence enhance <i>polyhedrin</i> expression in alphabaculoviruses: improvement of baculovirus expression vectors

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of the viral product polyhedrin (POLH), and the extremely high-level transcription of the <i>polh</i> gene has been exploited to express foreign proteins in insect cultured cells, larvae, and pupae. This <i>polh</i> hyper-transcription requires the "burst sequence" located between the transcriptional start site and the initiation codon. Here, we focused on the roles of the A-rich region within the burst sequence. We generated Bombyx mori nucleopolyhedrovirus mutants whose burst sequence contained "A-to-T" mutations in the A-rich region. Some mutants exhibited levels of <i>polh</i> promoter-driven reporter expression lower than or comparable to that of the wild type, whereas the mutants with TTT mutations at positions -16 to -14 in the <i>polh</i> upstream region showed a four- to fivefold increase in it. Most cases of single or double A-to-T mutations at -16 to -14 of the upstream region had small but significant effects on the expression level, while the triple mutation was the most effective. This enhancement was also observed in the Autographa californica multiple nucleopolyhedrovirus-based vector system, which is more commonly used for foreign protein expression. We also found that this triple mutation enhanced the accumulation of <i>polh</i> mRNA and POLH protein even in an OB-producing virus. These results indicate that specific mutations in the burst sequence have the potential to increase baculoviral protein expression at the transcriptional level and may improve foreign protein expression by baculoviruses.IMPORTANCEThe most notable characteristic of alphabaculoviruses is that they produce many proteinaceous occlusion bodies (OBs) during the very late stages of infection. The main component of these OBs is virus-encoded polyhedrin (POLH). The high expression of the <i>polh</i> gene led to the development of the baculovirus expression vector system (BEVS). Currently, this system is widely used for the production of vaccines, veterinary medicines, and reagents. Despite this background, the mechanisms by which baculoviruses ultimately produce large quantities of OBs remain largely unexplained, even after approximately 40 years since the BEVS development. Here, we discovered that three nucleotide substitutions in the <i>polh</i> burst sequence markedly increased the <i>polh</i> expression levels in both BmNPV- and AcMNPV-based BEVSs, regardless of the vector type. This discovery can be easily introduced into the currently used BEVS, possibly contributing to further improvements for achieving even higher expression of foreign proteins.