A seamless connection from the burst sequence to the start codon is essential for polyhedrin hyperexpression in alphabaculoviruses.

Alphabaculoviruses produce a large number of occlusion bodies (OBs) in host cells during the late stage of infection. OBs are mainly composed of polyhedrin (POLH), and high-level transcription of the polh gene has been exploited to express foreign proteins in insect cells. While making Bombyx mori nucleopolyhedrovirus (BmNPV) polh mutants using a conventional transfer vector-based method, we noticed that a virus with a short sequence insertion just before the polh start codon produces fewer very small OBs. Detailed analysis of several BmNPV mutants revealed that insertions between the burst sequence and start codon markedly decrease POLH accumulation and polh transcription. We further confirmed this decrease using recombinant viruses expressing a reporter gene driven by the polh promoter. These findings underscore the critical importance of a seamless connection from the burst sequence to the start codon for baculovirus polh hyperexpression.