Baculoviruses provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.

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Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two a-helices connected by a b-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

The pif gene (per os infectivity factor) of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) encodes a structural protein essential for oral infection. This protein is expressed in very low quantities. In this study, transcription and promoter analysis of SpliNPV pif were carried out to understand more fully the regulation of pif gene expression. Transcription in the pif gene region was examined using RT-PCR, Northern blot, primer extension, ribonuclease protection and 39 RACE. The pif gene was encoded by a late bicistronic messenger, which was characterized. This 1?9 kb messenger was present in very small amounts. In addition, this messenger was part of a set of six late mRNAs overlapping the pif sequence. A functional complementation assay was used to analyse the pif promoter. This assay allowed the detection of amounts of PIF which were sufficient for the production of orally infectious virions. The 13 bp region upstream from the initial ATG of pif was required and sufficient for the production of orally infectious virions. This promoter region was much shorter than the studied baculovirus promoters. A late promoter motif (TTAAG) is situated at the 59 end of this region. This motif was shown to be the promoter core by using single mutations of the motif in the complementation assay. These results suggest that the low expression of the pif gene is regulated chiefly at the transcriptional level.