Baculovirus Expression and Functional Analysis of Vpa2 Proteins from Bacillus thuringiensis

Abstract

The mode of action underlying the insecticidal activity of the Bacillus thuringiensis (Bt) binary pesticidal protein Vpa1/Vpa2 is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two novel Vpa2-like genes, Vpa2-like1 and Vpa2-like2, under the baculovirus p10 promoter in transfected Sf9 cells. Pairwise amino acid analyses revealed a higher percentage of identity and a lower number of gaps between Vpa2Ac1 and Vpa2-like2 than to Vpa2-like1. Moreover, Vpa2-like1 lacked the conserved Ser-Thr-Ser motif, involved in NAD binding, and the (F/Y)xx(Q/E)xE consensus sequence, characteristic of the ARTT toxin family involved in actin polymerization. Vpa2Ac1, Vpa2-like1 and Vpa2-like2 transcripts and proteins were detected in Sf9 culture cells, but the signals of Vpa2Ac1 and Vpa2-like2 were weak and decreased over time. Sf9 cells infected by a recombinant bacmid expressing Vpa2-like1 showed typical circular morphology and produced viral occlusion bodies (OBs) at the same level as the control virus. However, expression of Vpa2Ac1 and Vpa2-like2 induced cell polarization, similar to that produced by the microfilament-destabilizing agent cytochalasin D and OBs were not produced. The presence of filament disrupting agents, such as nicotinamide and nocodazole, during transfection prevented cell polarization and OB production was observed. We conclude that Vpa2Ac1 and Vpa2-like2 proteins likely possess ADP-ribosyltransferase activity that modulated actin polarization, whereas Vpa2-like1 is not a typical Vpa2 protein. Vpa2-like2 has now been designated Vpa2Ca1 (accession number AAO86513) by the Bacillus thuringiensis delta-endotoxin nomenclature committee.

Latent and Histopathological Impacts of Three Bioinsecticides on The Female Reproductive System of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae)

“…According to research by Harrison et al (2018), SpliNPV may be traced back to the genus Alphabaculovirus and the species Spodoptera littoralis nucleopolyhedrovirus. The main benefits of NPVs are their host specificity and lack of negative effects on beneficial insect and pollinator populations (Simón et al, 2020). However, the NPVs' limited use in pest control might be attributed to the lengthy incubation time required before they become lethal.…”

A Sensitive Immunodetection Assay Using Antibodies Specific to Staphylococcal Enterotoxin B Produced by Baculovirus Expression

“…Since BEVS is based on eukaryotic protein expression machinery and secretes recombinant proteins in a soluble form, proteins necessary for maintaining natural protein conformation have been produced with high purity and functionality [ 29 , 30 , 31 ]. To date, several bacterial and eukaryotic toxin molecules have been successfully purified using BEVS [ 31 , 32 , 33 , 34 ]; however, to the best of our knowledge, no studies have reported the use of BEVS to produce recombinant wild-type SEB.…”