Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins 英文参考文献.docVIP

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Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins 英文参考文献.doc

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Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins 英文参考文献

EfficientAssemblyandSecretionofRecombinant SubviralParticlesoftheFourDengueSerotypesUsing NativeprMandEProteins Pei-GangWang1*,MateuszKudelko1,JoanneLo2,4,LewisYuLamSiu1,KevinTszHinKwok1 ,Martin Sachse4,JohnM.Nicholls2,RobertoBruzzone1,RalfM.Altmeyer1,Be′atriceNal1,3 1HongKongUniversity-Pasteur Research Centre, TheUniversity ofHongKong,HongKong,China,2Department ofPathology, TheUniversity ofHongKong,Hong Kong, China, 3Department of Anatomy, The University of Hong Kong, Hong Kong, China, 4Plate-Forme de Microscopie Ultrastructurale, Institut Pasteur, Paris, France Abstract Background: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assemblyofprMandEenvelopeproteinsandarebelievedtoundergothesamematurationprocess.Denguerecombinant subviralparticleshavebeenproducedincellcultureswitheithermodifiedorchimericproteinsbutnotusingthenative formsofprMandE. Methodology/PrincipalFindings:Wehaveusedacodonoptimizationstrategytoobtainanefficientexpressionofnative viralproteinsandproductionofrecombinantsubviralparticles(RSPs)forallfourdenguevirus(DV)serotypes.AstableHeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similarmaturationprocessasdenguevirus.Pulsechaseexperimentshowedthat8hoursarerequiredforthesecretionof DV1RSPs.WehaveusedHeLa-prMEtodevelopasemi-quantitativeassayandscreenedahumansiRNAlibrarytargeting genesinvolvedinmembranetrafficking.Knockdownof23genesresultedinasignificantreductioninDVRSPsecretion, whereasfor22othersweobservedanincreaseofRSPlevelsincellsupernatant. Conclusions/Significance: Our data d