title: Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
authors.forename: Renée
authors.initials: R
authors.lastname: Lapointe
authors.affiliation: Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Wilson
authors.affiliation: Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
authors.forename: Lluïsa
authors.initials: L
authors.lastname: Vilaplana
authors.affiliation: Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
authors.forename: David R
authors.initials: DR
authors.lastname: O'Reilly
authors.affiliation: Dipartimento di Biologia, Difesa e Biotecnologie, Agro-Forestali-Università della Basilicata-Macchia Romana, 85100 Potenza, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Falabella
authors.affiliation: National Centre for Scientific Research 'Demokritos', 153 10 Aghia Paraskevi, Athens, Greece.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Douris
authors.affiliation: Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy (Québec), Canada G1V 4C7.
authors.forename: Michèle
authors.initials: M
authors.lastname: Bernier-Cardou
authors.affiliation: Dipartimento di Biologia, Difesa e Biotecnologie, Agro-Forestali-Università della Basilicata-Macchia Romana, 85100 Potenza, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pennacchio
authors.affiliation: National Centre for Scientific Research 'Demokritos', 153 10 Aghia Paraskevi, Athens, Greece.
authors.forename: Kostas
authors.initials: K
authors.lastname: Iatrou
authors.affiliation: Instituto di Genetica e Biofisica, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Malva
authors.affiliation: Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Olszewski
mesh_terms: D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007814:Larva; D007915:Lepidoptera / Q000502:physiology* / Q000821:virology; D018071:Polydnaviridae / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D018411:Spodoptera; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014863:Wasps / Q000821:virology*
chemical_list: D018076:DNA, Complementary; D014764:Viral Proteins