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Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells

Renée Lapointe^1,

Lluïsa Vilaplana^1,

David R. O'Reilly^1,

¹ Department of Biological Sciences, Imperial College, London SW7 2AZ, UK
² Dipartimento di Biologia, Difesa e Biotecnologie, Agro-Forestali-Università della Basilicata-Macchia Romana, 85100 Potenza, Italy
³ National Centre for Scientific Research ‘Demokritos’, 153 10 Aghia Paraskevi, Athens, Greece
⁴ Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy (Québec), Canada G1V 4C7
⁵ Instituto di Genetica e Biofisica, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy

Correspondence
Julie A. Olszewski
jaolsz{at}ship.edu

The polydnavirus Toxoneuron nigriceps bracovirus (TnBV) is an obligate symbiont associated with the braconid wasp T. nigriceps, a parasitoid of Heliothis virescens larvae. Previously, to identify polydnavirus genes that allow parasitization by altering the host immune and endocrine systems, expression patterns of TnBV genes from parasitized H. virescens larvae were analysed and cDNAs were obtained. To study the function of the protein from one such cDNA, TnBV1, overexpression of the protein was attempted by using the baculovirus Autographa californica multicapsid nucleopolyhedrovirus. Recovery of stable recombinant virus was unsuccessful, with the exception of recombinants with deletions/mutations within the TnBV1 gene. It was hypothesized that TnBV1 expression was cytotoxic to the Spodoptera frugiperda (Sf21) insect cells that were used to produce the recombinants. Therefore, the Bac-to-Bac system was used to create recombinant baculoviruses maintained in Escherichia coli expressing either TnBV1 (Ac-TnBV1) or an initiator-methionine mutant [Ac-TnBV1(ATG–)]. Microscopy revealed substantial cell death of Sf21 and High Five cells from 48 h post-infection with Ac-TnBV1, but not with the Ac-TnBV1(ATG–) recombinant virus. Ac-TnBV1-infected Sf21 cells, but not those with parental virus infection, showed an increased caspase-3-like protease activity, as well as increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) for breaks in host genomic DNA. Although indicative of apoptosis, blebbing and apoptotic bodies were not observed in infected cells. Transiently expressing TnBV1 alone caused TUNEL staining in High Five cells. These data suggest that TnBV1 expression alone can induce apoptosis-like programmed cell death in two insect cell lines. Injection of Ac-TnBV1 budded virus, compared with parental virus, did not result in an alteration of virulence in H. virescens larvae.

Present address: Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy (Québec), Canada G1V 4C7.

Present address: Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain.

Present address: Syngenta, Jealotts Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK.

||Present address: Biology Department, Shippensburg University of Pennsylvania, 1871 Old Main Drive, Shippensburg, PA 17257, USA.

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