Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em>
The mode of action underlying the insecticidal activity of the <i>Bacillus thuringiensis</i> (Bt) binary pesticidal protein Vpb/Vpa (formerly Vip1/Vip2) is uncertain. In this study, three recombinant baculoviruses were constructed using Bac-to-Bac technology to express Vpa2Ac1 and two no...
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MDPI AG
2020-08-01
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| Series: | Toxins |
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| Online Access: | https://www.mdpi.com/2072-6651/12/9/543 |
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| author | Oihane Simón Leopoldo Palma Ana Beatriz Fernández Trevor Williams Primitivo Caballero |
| author_facet | Oihane Simón Leopoldo Palma Ana Beatriz Fernández Trevor Williams Primitivo Caballero |
| author_sort | Oihane Simón |
| collection | DOAJ |
| description | The mode of action underlying the insecticidal activity of the <i>Bacillus thuringiensis</i> (Bt) binary pesticidal protein Vpb/Vpa (formerly Vip1/Vip2) 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 <i>Bacillus thuringiensis</i> delta-endotoxin nomenclature committee. |
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| language | English |
| last_indexed | 2024-03-10T16:59:43Z |
| publishDate | 2020-08-01 |
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| spelling | doaj.art-39b40fd29ba44e1fbc4a6edda861a1a82023-11-20T11:00:20ZengMDPI AGToxins2072-66512020-08-0112954310.3390/toxins12090543Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em>Oihane Simón0Leopoldo Palma1Ana Beatriz Fernández2Trevor Williams3Primitivo Caballero4Institute for Multidisciplinary Research in Applied Biology, Universidad Pública de Navarra, 31006 Pamplona, Navarra, SpainInstituto Académico Pedagógico de Ciencias Básicas y Aplicadas, Centro de Investigaciones y Transferencia de Villa María (CITVM-CONICET), Universidad Nacional De Villa María, Villa María, Córdoba 5900, ArgentinaInstitute for Multidisciplinary Research in Applied Biology, Universidad Pública de Navarra, 31006 Pamplona, Navarra, SpainInstituto de Ecología AC, Xalapa, Veracruz 91073, MexicoInstitute for Multidisciplinary Research in Applied Biology, Universidad Pública de Navarra, 31006 Pamplona, Navarra, SpainThe mode of action underlying the insecticidal activity of the <i>Bacillus thuringiensis</i> (Bt) binary pesticidal protein Vpb/Vpa (formerly Vip1/Vip2) 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 <i>Bacillus thuringiensis</i> delta-endotoxin nomenclature committee.https://www.mdpi.com/2072-6651/12/9/543vegetative insecticidal proteinsVpb/VpaentomopathogenADP-ribosyltransferaserecombinant baculoviruslepidopteran cells |
| spellingShingle | Oihane Simón Leopoldo Palma Ana Beatriz Fernández Trevor Williams Primitivo Caballero Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em> Toxins vegetative insecticidal proteins Vpb/Vpa entomopathogen ADP-ribosyltransferase recombinant baculovirus lepidopteran cells |
| title | Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em> |
| title_full | Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em> |
| title_fullStr | Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em> |
| title_full_unstemmed | Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em> |
| title_short | Baculovirus Expression and Functional Analysis of Vpa2 Proteins from <em>Bacillus thuringiensis</em> |
| title_sort | baculovirus expression and functional analysis of vpa2 proteins from em bacillus thuringiensis em |
| topic | vegetative insecticidal proteins Vpb/Vpa entomopathogen ADP-ribosyltransferase recombinant baculovirus lepidopteran cells |
| url | https://www.mdpi.com/2072-6651/12/9/543 |
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