Effect of signal sequence and promoter on the speed of action of a genetically modified Autographa californica nucleopolyhedrovirus expressing the scorpion toxin LqhIT2

Beek, Nikolai van; Lu, Albert; Presnail, J. K.; Davis, Dave; Greenamoyer, C. A.; Joraski, Kathleen; Moore, L. David; Pierson, Michelle; Herrmann, Robert O.; Flexner, Lindsey; Foster, James D.; Van, A. T.; Wong, Jeffrey A.; Jarvis, Donald L.; Hollingshaus, Gary; McCutchen, B. F.

doi:10.1016/s1049-9644(02)00172-x

Cited by 24 publications

(19 citation statements)

References 49 publications

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“…As previously observed for Bombyxin and EGT derived-signal peptides, the choice of signal sequence fused with an insect toxin resulted in significant effects for the virus pathology, mainly if the expression is controlled by an early promoter in insects [78,80]. Since the different ba3 gene variants were under the control of active late promoters, the difference in cell death between the immature and mature forms of the toxin was probably due to the compromising of the protein expression and secretion caused by the late viral infection.…”

Section: Discussionmentioning

confidence: 93%

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

et al. 2013

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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.

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Section: Discussionmentioning

confidence: 93%

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

et al. 2013

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“…Speed of action of genetically modified baculoviruses can be also enhanced by signal sequences in front of cloned genes. Van Beek et al (2003) constructed a series of AcMNPV recombinants expressing LqhIT2 scorpion toxin gene with different signal sequences, including signal sequences of AcMNPV GP64, cuticle protein II of Drosophila melanogaster, bombyxin of B. mori, dip-teran chymotrypsin and some scorpion toxins. Bombyxin signal sequence proved to be the most effective for enhancing insecticidal efficacy.…”

Section: Second Approach-genetic Modification Of Existing Baculovirusesmentioning

confidence: 99%

Baculoviruses — re-emerging biopesticides

Szewczyk

Hoyos-Carvajal

Paluszek

et al. 2006

Biotechnology Advances

236

161

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Biological control of agricultural pests has gained importance in recent years due to increased pressure to reduce the use of agrochemicals and their residues in the environment and food. Viruses of a few families are known to infect insects but only those belonging to the highly specialized family Baculoviridae have been used as biopesticides. They are safe to people and wildlife, their specificity is very narrow. Their application as bioinsecticides was limited until recently because of their slow killing action and technical difficulties for in vitro commercial production. Two approaches for the wider application of baculoviruses as biopesticides will be implemented in future. In countries where use of genetically modified organisms is restricted, the improvements will be mainly at the level of diagnostics, in vitro production and changes in biopesticide formulations. In the second approach, the killing activity of baculoviruses may be augmented by genetic modifications of the baculovirus genome with genes of another natural pathogen. It is expected that the baculoviruses improved by genetic modifications will be gradually introduced in countries which have fewer concerns towards genetically modified organisms.

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“…Studies employing different neurotoxins in which the effect of various promoters has been assayed for the speed of action, indicated that the effectiveness probably depended on the combination of promoter and toxin rather than on the promoter strength and timing alone (van Beek et al, 2003). The polyhedrin promoter, as a late and strong promoter, was selected to control the transcription of foreign neurotoxin genes RjAa17f or egfp.…”

Section: Discussionmentioning

confidence: 99%

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

Zhou²,

Meng³

et al. 2016

Insect Science

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A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV) expressing the insect-selective neurotoxin (RjAa17f) from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP-glucosyltransferase gene (egt) using λ-red homologous recombination system. Another egt deleted control HearNPV was constructed in a similar way by inserting egfp gene into the egt locus. One-step viral growth curve and viral DNA replication curve analysis confirmed that the recombination did not affect the viral growth and DNA replication in host cells. There is no discernable difference in occlusion-body morphogenesis between RjAa17f-HearNPV, Egfp-HearNPV and HZ8-HearNPV, which was confirmed by transmission electron microscopy analysis. However, the insecticidal activity of RjAa17f-HearNPV is enhanced against the third instar H. armigera larvae according to the bioassay on virulence comparison. There is a dramatic reduction (56.9%) in median lethal dose (LD ) and also a reduction (13.4%) in median survival time (ST ) for the recombinant RjAa17f-HearNPV compared to the HZ8-HearNPV, but only a 27.5% reduction in LD and 10.1% reduction in ST value when Egfp-HearNPV is compared with HZ8-HearNPV. The daily diet consumption analysis showed that the RjAa17f-HearNPV was able to inhibit the infected larvae feeding compared with the egt minus HearNPV. These results demonstrated that this novel recombinant RjAa17f-HearNPV could improve the insecticidal effect against its host insects and RjAa17f could be a considerable candidate for other recombinant baculovirus constructions.

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Effect of signal sequence and promoter on the speed of action of a genetically modified Autographa californica nucleopolyhedrovirus expressing the scorpion toxin LqhIT2

Cited by 24 publications

References 49 publications

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

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

Baculoviruses — re-emerging biopesticides

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

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