Abstract
Baculoviruses are the most studied insect viruses in the world and are used for biological control of agricultural and forest insect pests. They are also used as versatile vectors for expression of heterologous proteins. One of the major problems of their use as biopesticides is their slow speed to kill insects. Thus, to address this shortcoming, insect-specific neurotoxins from arachnids have been introduced into the baculovirus genome solely aiming to improve its virulence. In this work, an insecticide-like toxin gene was obtained from a cDNA derived from the venom glands of the theraphosid spider Brachypelma albiceps. The mature form of the peptide toxin (called Ba3) has a high content of basic amino acid residues, potential for three possible disulfide bonds, and a predicted three-stranded β-sheetDifferent constructions of the gene were engineered for recombinant baculovirus Autographa californica multiple nuclepolyhedrovirus (AcMNPV) expression. Five different forms of Ba3 were assessed; (1) the full-length sequence, (2) the pro-peptide and mature region, (3) only the mature region, and the mature region fused to an (4) insect or a (5) virus-derived signal peptide were inserted separately into the genome of the baculovirus. All the recombinant viruses induced cell death by necrosis earlier in infection relative to a control virus lacking the toxin gene. However, the recombinant virus containing the mature portion of the toxin gene induced a faster cell death than the other recombinants. We found that the toxin construct with the signal peptide and/or pro-peptide regions delayed the necrosis phenotype. When infected cells were subjected to ultrastructural analysis, the cells showed loss of plasma membrane integrity and structural changes in mitochondria before death. Our results suggest this use of baculovirus is a potential tool to help understand or to identify the effect of insect-specific toxic peptides when produced during infection of insect cells.
Highlights
Insects are a major cause of reduction in crop yields and currently chemical insecticides are still the dominant method for controlling pest populations [1]
Spider venom peptides are commonly rich in disulfide bonds and have been found to be one of the major contributors to the insecticidal activity [12]
Based on the information obtained from direct peptide sequencing of Ba1 [33], a specific oligonucleotide (Table S1) was designed and used for the PCR reaction using the cDNA library as a template
Summary
Insects are a major cause of reduction in crop yields and currently chemical insecticides are still the dominant method for controlling pest populations [1]. Due to the negative environmental impact of chemical insecticides and the appearance of resistant insects, the search for alternative methods of controlling insect pests has increased. Biological control methods such as insecticidal toxins, present in many venomous organisms [2,3] have been shown to be a reasonable option for replacing chemical agents [4]. Spider venom peptides are commonly rich in disulfide bonds and have been found to be one of the major contributors to the insecticidal activity [12]. The low viability of venom and the difficulty of peptide purification have hampered application of insecticidal peptides in insect pest control [12]
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