Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells.
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.
Lapointe
Renée
R
Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
Wilson
Rebecca
R
Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
Vilaplana
Lluïsa
L
Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
O'Reilly
David R
DR
Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
Falabella
Patrizia
P
Dipartimento di Biologia, Difesa e Biotecnologie, Agro-Forestali-Università della Basilicata-Macchia Romana, 85100 Potenza, Italy.
Douris
Vassilis
V
National Centre for Scientific Research 'Demokritos', 153 10 Aghia Paraskevi, Athens, Greece.
Bernier-Cardou
Michèle
M
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, Sainte-Foy (Québec), Canada G1V 4C7.
Pennacchio
Francesco
F
Dipartimento di Biologia, Difesa e Biotecnologie, Agro-Forestali-Università della Basilicata-Macchia Romana, 85100 Potenza, Italy.
Iatrou
Kostas
K
National Centre for Scientific Research 'Demokritos', 153 10 Aghia Paraskevi, Athens, Greece.
Malva
Carla
C
Instituto di Genetica e Biofisica, CNR, Via Pietro Castellino 111, 80131 Napoli, Italy.
Olszewski
Julie A
JA
Department of Biological Sciences, Imperial College, London SW7 2AZ, UK.
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*
D018076:DNA, Complementary; D014764:Viral Proteins