A New theraphosid Spider Toxin Causes Early Insect Cell Death by Necrosis When Expressed In Vitro during Recombinant Baculovirus Infection

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.

Improved insecticidal activity of a recombinant baculovirus expressing spider venom cyto-insectotoxin

“…Cells infected with BmNPV/Polh-Cit1a seemed to lose its cytoplasm and outline of cells was blurred (data not shown). This result was similar to the previous paper that a theraphosid spider toxin caused early Sf21 cell death by necrosis when this peptide toxin was expressed using recombinant AcMNPV (Ardisson-Araújo et al 2013). …”

Recombinant Helicoverpa armigera nucleopolyhedrovirus with arthropod‐specific neurotoxin gene RjAa17f from Rhopalurus junceus enhances the virulence against the host larvae

“…A plausible explanation might be associated with the accumulation of RjAa17f that likely destroyed the cell structure, thereby reducing the production of BV. Similarly, scorpion neurotoxin BmK Mm2 from Buthus martensii could induce the apoptosis of glial cells (Fu et al ., ), and recombinant viruses containing the pro‐peptide, or mature region of insect‐specific neurotoxins from arachnids induced cell death by necrosis earlier in infection (Ardisson‐Araujo et al ., ).…”

Screening and Validation of Highly-Efficient Insecticidal Conotoxins from a Transcriptome-Based Dataset of Chinese Tubular Cone Snail

“…Genetic engineering has been applied to modify the insecticidal activity of baculoviruses, and presents a great elevation to improve the effectiveness of insect viruses [ 34 ]. The main purpose of realizing genetic modification of wild-type baculoviruses by inserting toxin fragment(s) is to increase the effectiveness and speed to kill target insect pests.…”