Article: Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins
| Title | Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Authors | Wang, PG3 Kudelko, M3 Lo, J1 2 Yu Lam Siu, L3 Tsz Hin Kwok, K3 Sachse, M2 Nicholls, JM1 Bruzzone, R3 Altmeyer, RM3 Nal, B1 3 | ||||||||||
| Issue Date | 2009 | ||||||||||
| Publisher | Public 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?] DOI: http://dx.doi.org/10.1371/journal.pone.0008325 | ||||||||||
| Abstract | Background: 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. | ||||||||||
| ISSN | 1932-6203 2011 Impact Factor: 4.092 2011 SCImago Journal Rankings: 0.519 | ||||||||||
| DOI | http://dx.doi.org/10.1371/journal.pone.0008325 | ||||||||||
| ISI Accession Number ID | WOS:000272833800018
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. | ||||||||||
| PubMed Central ID | PMC2790604 | ||||||||||
| References | References in Scopus |
| dc.contributor.author | Wang, PG | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| dc.contributor.author | Kudelko, M | ||||||||||
| dc.contributor.author | Lo, J | ||||||||||
| dc.contributor.author | Yu Lam Siu, L | ||||||||||
| dc.contributor.author | Tsz Hin Kwok, K | ||||||||||
| dc.contributor.author | Sachse, M | ||||||||||
| dc.contributor.author | Nicholls, JM | ||||||||||
| dc.contributor.author | Bruzzone, R | ||||||||||
| dc.contributor.author | Altmeyer, RM | ||||||||||
| dc.contributor.author | Nal, B | ||||||||||
| dc.date.accessioned | 2011-03-28T09:28:31Z | ||||||||||
| dc.date.available | 2011-03-28T09:28:31Z | ||||||||||
| dc.date.issued | 2009 | ||||||||||
| dc.description.abstract | Background: 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.nature | published_or_final_version | ||||||||||
| dc.identifier.citation | Plos One, 2009, v. 4 n. 12 [How to Cite?] DOI: http://dx.doi.org/10.1371/journal.pone.0008325 | ||||||||||
| dc.identifier.doi | http://dx.doi.org/10.1371/journal.pone.0008325 | ||||||||||
| dc.identifier.epage | e8325 | ||||||||||
| dc.identifier.hkuros | 172049 | ||||||||||
| dc.identifier.isi | WOS:000272833800018
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.issn | 1932-6203 2011 Impact Factor: 4.092 2011 SCImago Journal Rankings: 0.519 | ||||||||||
| dc.identifier.issue | 12 | ||||||||||
| dc.identifier.pmcid | PMC2790604 | ||||||||||
| dc.identifier.pmid | 20016834 | ||||||||||
| dc.identifier.scopus | eid_2-s2.0-77954066120 | ||||||||||
| dc.identifier.spage | e8325 | ||||||||||
| dc.identifier.uri | http://hdl.handle.net/10722/132718 | ||||||||||
| dc.identifier.volume | 4 | ||||||||||
| dc.language | eng | ||||||||||
| dc.publisher | Public Library of Science. The Journal's web site is located at http://www.plosone.org/home.action | ||||||||||
| dc.publisher.place | United States | ||||||||||
| dc.relation.ispartof | PLoS ONE | ||||||||||
| dc.relation.references | References in Scopus | ||||||||||
| dc.rights | Creative Commons: Attribution 3.0 Hong Kong License | ||||||||||
| dc.subject.mesh | Codon - genetics | ||||||||||
| dc.subject.mesh | Dengue Virus - classification - genetics - physiology | ||||||||||
| dc.subject.mesh | Viral Proteins - genetics - metabolism | ||||||||||
| dc.subject.mesh | Virion - metabolism | ||||||||||
| dc.subject.mesh | Virus Assembly - physiology | ||||||||||
| dc.title | Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins | ||||||||||
| dc.type | Article |
Author Affiliations
- The University of Hong Kong
- Institut Pasteur, Paris
- HKU-Pasteur Research Centre