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Article: Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins

TitleEfficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins
Authors
Issue Date2009
PublisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.action
Citation
Plos One, 2009, v. 4 n. 12 How to Cite?
AbstractBackground: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. Methodology/Principal Findings: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa 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 similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. Conclusions/Significance: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus. © 2009 Wang et al.
Persistent Identifierhttp://hdl.handle.net/10722/132718
ISSN
2014 Impact Factor: 3.234
2014 SCImago Journal Rankings: 1.300
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
6th European Framework programme DENFRAME
Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID)08070952
University of Hong Kong
Consulate General of France in Hong Kong
Funding Information:

This work was supported by the 6th European Framework programme DENFRAME and by the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070952). MK is a PhD student supported by the University of Hong Kong. JL is an MPhil student supported by the University of Hong Kong, and by the "Alexandre Yersin Excellence Scholarship" awarded by the Consulate General of France in Hong Kong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

 

DC FieldValueLanguage
dc.contributor.authorWang, PGen_HK
dc.contributor.authorKudelko, Men_HK
dc.contributor.authorLo, Jen_HK
dc.contributor.authorYu Lam Siu, Len_HK
dc.contributor.authorTsz Hin Kwok, Ken_HK
dc.contributor.authorSachse, Men_HK
dc.contributor.authorNicholls, JMen_HK
dc.contributor.authorBruzzone, Ren_HK
dc.contributor.authorAltmeyer, RMen_HK
dc.contributor.authorNal, Ben_HK
dc.date.accessioned2011-03-28T09:28:31Z-
dc.date.available2011-03-28T09:28:31Z-
dc.date.issued2009en_HK
dc.identifier.citationPlos One, 2009, v. 4 n. 12en_HK
dc.identifier.issn1932-6203en_HK
dc.identifier.urihttp://hdl.handle.net/10722/132718-
dc.description.abstractBackground: Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E. Methodology/Principal Findings: We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa 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 similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant. Conclusions/Significance: Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus. © 2009 Wang et al.en_HK
dc.languageengen_US
dc.publisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.actionen_HK
dc.relation.ispartofPLoS ONEen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.meshCodon - genetics-
dc.subject.meshDengue Virus - classification - genetics - physiology-
dc.subject.meshViral Proteins - genetics - metabolism-
dc.subject.meshVirion - metabolism-
dc.subject.meshVirus Assembly - physiology-
dc.titleEfficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteinsen_HK
dc.typeArticleen_HK
dc.identifier.emailNicholls, JM: jmnichol@hkucc.hku.hken_HK
dc.identifier.emailBruzzone, R: bruzzone@hkucc.hku.hken_HK
dc.identifier.emailNal, B: bnal@hkucc.hku.hken_HK
dc.identifier.authorityNicholls, JM=rp00364en_HK
dc.identifier.authorityBruzzone, R=rp01442en_HK
dc.identifier.authorityNal, B=rp00541en_HK
dc.description.naturepublished_or_final_versionen_US
dc.identifier.doi10.1371/journal.pone.0008325en_HK
dc.identifier.pmid20016834en_HK
dc.identifier.pmcidPMC2790604-
dc.identifier.scopuseid_2-s2.0-77954066120en_HK
dc.identifier.hkuros172049-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-77954066120&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume4en_HK
dc.identifier.issue12en_HK
dc.identifier.spagee8325en_HK
dc.identifier.epagee8325en_HK
dc.identifier.isiWOS:000272833800018-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridWang, PG=7405460758en_HK
dc.identifier.scopusauthoridKudelko, M=35083703200en_HK
dc.identifier.scopusauthoridLo, J=35748856900en_HK
dc.identifier.scopusauthoridYu Lam Siu, L=36144833100en_HK
dc.identifier.scopusauthoridTsz Hin Kwok, K=36144407600en_HK
dc.identifier.scopusauthoridSachse, M=6603781368en_HK
dc.identifier.scopusauthoridNicholls, JM=7201463077en_HK
dc.identifier.scopusauthoridBruzzone, R=7006793327en_HK
dc.identifier.scopusauthoridAltmeyer, RM=7003677186en_HK
dc.identifier.scopusauthoridNal, B=6506672380en_HK

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