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Article: Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins
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TitleEfficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins
 
AuthorsWang, PG3
Kudelko, M3
Lo, J1 2
Yu Lam Siu, L3
Tsz Hin Kwok, K3
Sachse, M2
Nicholls, JM1
Bruzzone, R3
Altmeyer, RM3
Nal, B3 1
 
Issue Date2009
 
PublisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.action
 
CitationPlos One, 2009, v. 4 n. 12 [How to Cite?]
DOI: http://dx.doi.org/10.1371/journal.pone.0008325
 
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.
 
ISSN1932-6203
2012 Impact Factor: 3.73
2012 SCImago Journal Rankings: 1.512
 
DOIhttp://dx.doi.org/10.1371/journal.pone.0008325
 
PubMed Central IDPMC2790604
 
ISI Accession Number IDWOS:000272833800018
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.

 
ReferencesReferences in Scopus
 
DC FieldValue
dc.contributor.authorWang, PG
 
dc.contributor.authorKudelko, M
 
dc.contributor.authorLo, J
 
dc.contributor.authorYu Lam Siu, L
 
dc.contributor.authorTsz Hin Kwok, K
 
dc.contributor.authorSachse, M
 
dc.contributor.authorNicholls, JM
 
dc.contributor.authorBruzzone, R
 
dc.contributor.authorAltmeyer, RM
 
dc.contributor.authorNal, B
 
dc.date.accessioned2011-03-28T09:28:31Z
 
dc.date.available2011-03-28T09:28:31Z
 
dc.date.issued2009
 
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.
 
dc.description.naturepublished_or_final_version
 
dc.identifier.citationPlos One, 2009, v. 4 n. 12 [How to Cite?]
DOI: http://dx.doi.org/10.1371/journal.pone.0008325
 
dc.identifier.doihttp://dx.doi.org/10.1371/journal.pone.0008325
 
dc.identifier.epagee8325
 
dc.identifier.hkuros172049
 
dc.identifier.isiWOS:000272833800018
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.

 
dc.identifier.issn1932-6203
2012 Impact Factor: 3.73
2012 SCImago Journal Rankings: 1.512
 
dc.identifier.issue12
 
dc.identifier.pmcidPMC2790604
 
dc.identifier.pmid20016834
 
dc.identifier.scopuseid_2-s2.0-77954066120
 
dc.identifier.spagee8325
 
dc.identifier.urihttp://hdl.handle.net/10722/132718
 
dc.identifier.volume4
 
dc.languageeng
 
dc.publisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.action
 
dc.publisher.placeUnited States
 
dc.relation.ispartofPLoS ONE
 
dc.relation.referencesReferences in Scopus
 
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 proteins
 
dc.typeArticle
 
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Author Affiliations
  1. The University of Hong Kong
  2. Institut Pasteur, Paris
  3. HKU-Pasteur Research Centre