Effect of signal sequence and promoter on the speed of action of a genetically modified Autographa californica nucleopolyhedrovirus expressing the scorpion toxin LqhIT2

We report on the construction and optimization of recombinant Autographa californica nucleopolyhedrovirus engineered to express the insect-selective toxin IT2 from the scorpion Leiurus quinquestriatus hebraeus. We constructed a series of viruses expressing the synthetic Lqhit2 gene with different signal sequences or controlled by different promoters. The effect of the various viruses on speed of response was assayed in Heliothis virescens larvae. In addition, the performance of the optimum recombinant viral construct was compared with similar constructs carrying the Androctonus australis hector insect toxin controlled by two different promoters. There were no significant differences in speed of response of viruses with the early hr5/ie1 the early hr5/lef3, or the early/late hr5/39Kpromoter driving toxin expression. However, the choice of signal sequence resulted in significant effects. The signal sequence from the bombyxin gene of the silkworm Bombyx mori, proved nominally the best. When the signal sequences were used from the following genes, the viruses acted significantly slower: AcMNPV gp67, a lepidopteran adipokinetic hormone, a dipteran chymotrypsin, and the homologous Lqhit2. Finally, the signal sequences of the genes for cuticle protein II of Drosophila melanogaster and of the insect toxins of the scorpions A. australis hector and Hottentota judaicus performed very poorly. The speed of action of AcMNPV, carrying the synthetic Lqhit2 gene with the bombyxin secretion signal and driven by the hr5/ie1 promoter [Ac.LqhIT2(hr5/ie1)], was compared to that of the same virus carrying the AaIT gene under the control of the p10 promoter, or the hr5/ie1 promoter in H. virescens, Trichoplusia ni, and Spodoptera exigua larvae. All recombinant viruses elicited the response significantly faster than the common progenitor wild-type virus in all tests. The response elicited by Ac.LqhIT2(hr5/ie1) was nominally faster than that of both viruses expressing AaIT in all insects tested.