Functional expression of an alpha anti‐insect scorpion neurotoxin in insect cells and lepidopterous larvae

Chejanovsky, Nor; Zilberberg, Noam; Rivkin, Hadasah; Zlotkin, Eliahu; Gurevitz, Michael

doi:10.1016/0014-5793(95)01263-4

Cited by 57 publications

(42 citation statements)

References 10 publications

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“…The percent mortality was calculated as the number of dead larvae (excluding larvae killed by the injection, normally one or two) divided by the number of larvae that survived the infection. No mortality was observed for mock-infected larvae (12,23).…”

Section: Methodsmentioning

confidence: 97%

Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells

Chejanovsky

2003

J Virol

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Infection of Spodoptera littoralis SL2 cells with the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) results in apoptosis and low yields of viral progeny, in contrast to infection with S. littoralis nucleopolyhedrovirus (SlNPV). By cotransfecting SL2 cells with AcMNPV genomic DNA and a cosmid library representing the complete SlNPV genome, we were able to rescue AcMNPV replication and to isolate recombinant virus vAcSL2, which replicated efficiently in SL2 cells. Moreover, vAcSL2 showed enhanced infectivity for S. littoralis larvae compared to AcMNPV. The genome of vAcSL2 carried a 519-bp insert fragment that increased the distance between the TATA element and the transcriptional initiation site (CAGT) of immediate-early gene ie0. This finding correlated with low steady-state levels of IE0 and higher steady-state levels of IE1 (the product of the ie1 gene, a major AcMNPV transactivator, and a multifunctional protein) than of IE0. Mutagenesis of the ie0 promoter locus by insertion of the chloramphenical acetyltransferase (cat) gene yielded a new recombinant AcMNPV with replication properties identical to those of vAcSL2. Thus, the analysis indicated that increasing the steady-state levels of IE1 relative to IE0 should enable AcMNPV replication in SL2 cells. This suggestion was confirmed by constructing a recombinant AcMNPV bearing an extra copy of the ie1 gene under the control of the Drosophila hsp70 promoter. These results suggest that IE0 plays a role in the regulation of AcMNPV infection and show, for the first time, that significant improvement in the ability of AcMNPV to replicate in a poorly permissive cell line and organism can be achieved by increasing the expression of the main multiple functional protein, IE1.The arthropod virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the Baculoviridae family, is able to infect 32 lepidopteran species (24). The complete genome of AcMNPV has been sequenced (3), and its replication in permissive cells has been studied extensively (7,33). AcMNPV vectors are widely used as efficient tools for the expression of "foreign genes" in established cell lines and whole insects (7, 53). Recombinant AcMNPVs have been developed for use in pest control (29, 31, 48); moreover, baculovirus-based vectors have been considered for gene therapy purposes (1, 17). The ability of AcMNPV to interact with a wide range of hosts (50) makes it an attractive tool for elucidating which molecular factors are involved in determination of the host range of baculoviruses. Since AcMNPV can penetrate the nuclei of nonpermissive insect and mammalian cells, important mechanisms that determine the host range of AcM-NPV are related to the expression of the viral genome (20,44,(49)(50)(51).AcMNPV gene expression is divided into early, late, and very late phases. Early-phase genes are transcribed by host RNA polymerase II before DNA replication, while late-and very-late-phase genes depend on viral DNA replication and are transcribed by a virus-specifi...

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Section: Methodsmentioning

confidence: 97%

Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells

Chejanovsky

2003

J Virol

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“…This enhancement of the insecticidal activity is achieved by engineering nucleopolyhedroviruses in such a way that their natural insect pathogenicity is combined with the expression of insect-selective neurotoxins . As such a 30-40% improved insecticidal activity is achieved (Chejanovsky et al, 1995). The gene encoding for a insect-selective neurotoxin is subcloned into a transfer vector plasmid, which is then cotransfected with the parental baculovirus DNA into an appropriate insect cell line so that the neurotoxin encoding gene is transferred to the baculovirus by homologous recombination (Zlotkin et al, 2000).…”

Section: Incorporation Of Insect-selective Toxins In Recombinant Bacumentioning

confidence: 99%

The Sophisticated Peptide Chemistry of Venomous Animals as a Source of Novel Insecticides Acting on Voltage-Gated Sodium Channels

Peigneur¹,

Tytgat²

2012

Insecticides - Advances in Integrated Pest Management

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“…Many reports have shown success in improving the insecticidal effectiveness of baculoviruses, either through the expression of insecticidal proteins, neurotoxins or juvenile hormone inhibitors (Carbonell et al, 1988 ;Merryweather et al, 1990 ;Tomalski & Miller, 1991 ;Stewart et al, 1991 ;Maeda et al, 1991 ;Tomalski et al, 1993 ;Ribeiro & Crook, 1993 ;Cory et al, 1994 ;Chejanovsky et al, 1995 ;Eldridge et al, 1995 ;Heinz et al, 1995). However, concerns remain over possible effects on non-target organisms as well as acquired insect resistance to introduced foreign proteins.…”

Section: The Antisense Strategy Applied To Insect Pest Controlmentioning

confidence: 99%

“…Success in this direction has been achieved through the introduction of genes encoding bacterial or arthropod insectselective toxins or juvenile hormone inhibitors (Carbonell et al, 1988 ;Merryweather et al ;1990 ;Tomalski & Miller, 1991 ;Stewart et al, 1991 ;Maeda et al, 1991 ;Tomalski et al, 1993 ;Ribeiro & Crook, 1993 ;Cory et al, 1994 ;Chejanovsky et al, 1995 ;Eldridge et al, 1995 ;Heinz et al, 1995). However, concerns remain over target selectivity and the possibility of development of insect resistance to the foreign protein, as illustrated in the case of the Bacillus thuringiensis toxin (McGaughey, 1994).…”

Section: Introductionmentioning

confidence: 99%

Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.

Lee

Chen

et al. 1997

Journal of General Virology

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Attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. However, it is often difficult to ensure correct processing or secretion of the encoded peptides. Here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. We selected the c-myc gene for two main reasons : (i) its protein is known to be well conserved in evolution and to have multiple essential functions during development ; and (ii) c-myc family genes have yet to be characterized in insects, thus blockage of essential genes by antisense transcripts from a strong virus promoter could provide a sensitive test for the existence of myc-like gene products. An appropriate fragment of the

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Functional expression of an alpha anti‐insect scorpion neurotoxin in insect cells and lepidopterous larvae

Abstract: with wild type AcNPV. The present expression system will facilitate: (1) the future elucidation of structural elements involved in its prominent anti-insect toxicity; and (2) the future design of genetically modified alpha toxins with improved anti-insect selectivity.

Cited by 57 publications

References 10 publications

Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells

Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells

The Sophisticated Peptide Chemistry of Venomous Animals as a Source of Novel Insecticides Acting on Voltage-Gated Sodium Channels

Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.

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