INSTITUTO DE BIOTECNOLOGIA Y BIOLOGIA MOLECULAR
Unidad Ejecutora - UE
congresos y reuniones científicas
Recombinant polyhedron envelope protein is able to package different species of wild type Baculovirus occlusion bodies upon infection of a stably transformed UFLAg-286 cell line
PIDRE, MATÍAS LUIS; FABRE, MARÍA LAURA; ROMANOWSKI, VÍCTOR; MASSON, TOMAS
Congreso; XV Congreso de la Sociedad Española de Virología (SEV); 2019
IntroductionSomebaculoviruses are used as biopesticides to control lepidopteranpests. Anticarsiagemmatalis multiplenucleopolyhedrovirus (AgMNPV) has been a successful example in warmareas of Latin America. However, its performance in more temperateclimates requires improvement to increase its speed of action orvirulence. To this end we set out to explore the inclusion ofrecombinant polypeptides in the occlusion bodies (OBs) or polyhedraof the AgMNPV that initiate the oral infection of susceptible larvae.To proof this concept we generated an insect cell line expressing theenhanced green fluorescent protein (eGFP) fused to the polyhedronenvelope protein (PEP) of AgMNPV and evaluated its inclusion in theOBs produced after infection with two wild type baculoviruses.Material& MethodsTogenerate the expression plasmid pIP-GFP::PEPAg,theORF encoding AgMNPVPEP wasamplifiedby PCR and cloned into pIP-GFP in frame with gfpORF. UFLAg-286 cells were seeded in a 6-well plate (2 × 106per well) and transfected with 1 μg of plasmid pIP-GFP::PEPAgusing 3μl of polyethyleneimine (PEI) reagent. UFLAg cells stablyexpressing GFP::PEPAgwere selected with puromycin for 15 days and then cloned using theterminal dilution method. Monoclonal transgenic cell lines wereinfected with AgMNPV. Purified OBs were analyzed by confocalmicoscopy and scanning electron microscopy (12,5 kV, FEI Quanta 200).OBs mass spectrometry analyses were conducted by nanoLC-MS/MS in aThermo Scientific Q Exactive Mass Spectrometer coupled to a nanoHPLCEASY-nLC 1000. ResultsToevaluate the expression and localization of the GFP::PEPAgprotein in the OBs, UFLAg cells expressing GFP::PEPAg and UFLAg control cells (expressing GFP ) were infected with wtAgMNPV and AcMNPV. Confocal micorscopy analysis, showed GFPfluorescence indicating the presence of GFP::PEPAgin the OB envelope of both viruses. SEM analysis showed that AgMNPVand AcMNPV OBs had normal morphology. Finally, proteomics dataindicated that GFP::PEPAgwas present in both OBs, although the packaging in the homologousAgMNPV was more efficient than in the heterologous AcMNPV. DiscussionProteomicand microscopy techniques confirmed that a single susceptible insectcell line expressing a recombinant PEP has a powerful capacity totrap chimeric proteins within the envelope of AgMNPV OBs. Inaddition, it is was able to surround the occlusion body of wild typeAcMNPV. This proves that PEP chimaeras can be loaded onto wild typeOBs and brings about the possibility of testing alternativepolypeptides to facilitate the entry of biopesticidal baculovirusesto the midgut epithelium of the target pest and or releasing specifictoxic proteins immediately after infection. This strategy avoids therelease of genetically modified baculoviruses in the environmentwhich is a major concern for state regulatory agencies.