Full-length (72K) and truncated (61K) CryIVD mosquitocidal proteins of (Bt) were expressed in cells and larvae of using a baculovirus vector to investigate the role of CryIVD peptides in toxicity as well as to evaluate further the baculovirus/lepidopteran system for expressing Bt proteins. The genes were inserted into the multinucleocapsid nuclear polyhedrosis virus (AcMNPV) under control of the polyhedrin promoter by recombination in cells between a transfer vector carrying the Bt genes and vDA26Z, a recombinant AcMNPV carrying the β-galactosidase gene under control of the DA26 promoter. Recombinant AcMNPVs carrying the genes were detected as blue occlusion body-negative plaques in monolayers of cells grown in the presence of X-Gal. Infection of cells and larvae with plaque-purified recombinant virus, expressing either the full-length or truncated CryIVD protein, resulted in the synthesis of proteins of the expected size, as confirmed by immunoblot analyses, and their crystallization into cuboidal inclusions in the cytoplasm. Infected cells and purified inclusions from the virus (AcCryIVD) expressing the full-length protein were highly toxic to mosquito larvae, but similar preparations from the virus (AcCryIVD-C) expressing the truncated protein with a 9.6K deletion at the N terminus were non-toxic. Proteolysis with trypsin of CryIVD proteins produced by Bt and the recombinant AcMNPVs yielded peptides corresponding in size, showing that synthesis of mosquitocidal Bt proteins in lepidopteran cells occurred. The lack of toxicity of the truncated CryIVD protein, which like the toxic full-length protein yielded a 34K protein on proteolysis that has been implicated in toxicity, indicates that by itself this protein is non-toxic. These results demonstrate the utility of the baculovirus system for expression of mosquitocidal Bt proteins and for investigation of their mode of action.


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