Their genome encodes a polyprotein precursor of at least seven non-structural proteins and three structural proteins which are the capsid protein (C), the membrane protein (M) and the envelope glycoprotein (E). There are four serotypes of dengue virus (DV1-4). Therefore, there is an urgent need to develop new tools to gain insight into the viral journey through host cells.Īs a member of the Flavivirus genus in the Flaviridae family, DV is a small, positive strand RNA enveloped virus. However, the interaction between dengue virus (DV) and host cells is only partly understood. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.ĭengue is one of the most important vector-borne viral diseases in humans. 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. MK is a PhD student supported by the University of Hong Kong. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: 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). Received: AugAccepted: NovemPublished: December 15, 2009Ĭopyright: © 2009 Wang et al.
PLoS ONE 4(12):Įditor: Maciej Lesniak, The University of Chicago, United States of America (2009) Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins. 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.Ĭitation: Wang P-G, Kudelko M, Lo J, Siu LYL, Kwok KTH, Sachse M, et al. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. 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. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed.
A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. 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.
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