BACULOVIRUS EXPRESSION OF AN INSECT GENE THAT ENCODES MULTIPLE NEUROPEPTIDES

Sex pheromone production in the corn earworm, Helicoverpa ten, is regu lated by a 33-amino-acid neuropeptide named Hez-PBAN (pheromone biosyn thesis activating neuropeptide). Hez-PBAN is encoded in a preprohormon e that also contains four other structurally related peptides. Two rec ombinant baculoviruses that contain two different sequences of Hez-PBA N cDNA under the control of a strong polyhedrin promotor were construc ted. The first virus, AcWT-PBAN, contains the entire prepro-Hez-PBAN c oding sequence. The second virus, AcBX-PBAN, contains a synthetic chim era gene encoding a bombyxin signal peptide sequence fused to a pro-He z-PBAN sequence. Cell extracts, culture medium of BTI-TN-5B 1-4 cells, and hemolymph from 4th instar Trichoplusia ni larvae, all infected wi th AcBX-PBAN, showed a high level of pheromonotropic activity. Pheromo notropic activity was not detected in the cells infected with AcWT-PBA N. Results of chromatographic and immunochemical studies showed that s ome of the potential processing sites in the expressed pro-Hez-PBAN se quence were not used during posttranslational processing in the AcBX-P BAN-4-infected BTI-TN-5BI-4 cells and 4th instar T. ni larvae. However , the processing pattern of the recombinant pro-Hez-PBAN in AcBX-PBAN- infected 4th instar T. ni larvae was similar to that exhibited in the central nervous system of H. zea adult females, since a PBAN-like immu noreactive-peptide-band was found in the hemolymph of Ac-BX-PBAN-4-inf ected 4th instar T. ni larvae. In a droplet feeding assay, neonate and 3rd instar T. ni larvae infected with AcBX-PBAN-4 showed a significan t reduction in survival time (26% and 19%, respectively) when compared to control larvae that were infected with a polyhedrin-deficient viru s, Ac-E10. (C) 1998 Elsevier Science Ltd. All rights reserved.