In this study, we have used a codon-optimization strategy to establish and characterize stable cell lines that produce RSPs for the four dengue serotypes. Previous studies had reported the production of DV RSPs in which the glycoproteins were mutated either to avoid host cell fusion or to delete the ER retention signal. For example, DV1 RSPs were not secreted effectively and DV2 RSPs could not form unless the transmembrane domain of the E glycoprotein, which contains an ER retention motif was replaced with that of JEV [22, 23, 34] . Our ability to obtain the efficient production of RSPs of all serotypes without any change in the amino acid sequence is likely the result of codon optimization for expression in mammalian cells, which has been proven to be an Figure 6 . Sucrose gradient analysis of DV1 RSPs. The supernatant from HeLa-prME cells was concentrated and resuspended in PBS or 0.5% Triton-X 100 containing PBS. RSPs were then centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm (Beckman SW-41Ti rotor) for 2.5 hours at 4uC. Fractions of 0.5 ml were collected and E content was measured using CLDB. The percentage of E protein in each fraction is displayed on the Y axis. doi:10.1371/journal.pone.0008325.g006 Figure 7 . Application of the DV RSP-producing HeLa-prME cell line and CLDB to screen a small library of siRNA which targets 122 genes involved in membrane trafficking. A) The correlation between luminescence density and amount of E protein on PVDF membrane in CLDB assay. B) The screen results of the siRNA library which targets 122 genes involved in membrane trafficking. Non-targeting siRNA (NT) and siRNA targeting DV1 prME were used as controls. Level of E protein in cell supernatant of each siRNA was expressed as its ratio to that of NT controls. Two-sided Student's t test was used to assess the statistical significance of differences between each sample and NT. Differences were considered statistically significant when P,0.05. Genes inducing either a significant decrease or increase in RSPs production are shown in gray and black columns, respectively. doi:10.1371/journal.pone.0008325.g007 , respectively (B) . C) Production of RSPs by 293T-prME and HeLa-prME stable cell lines. DV1-DV3 RSPs in supernatants were analyzed by SDS-PAGE and silver staining of polyacrylamide gels. Bands corresponding to the approximate molecular weight of E monomers and dimers, as well as prM and M are indicated by arrows. Supernatants from parental 293T and HeLa cells were used as controls. D) Analysis of RSPs of 4 serotypes by sucrose gradient. RSPs were centrifuged in a 20 to 60% sucrose gradient at 28,000 rpm for 2.5 hours in 4uC. Fractions of 0.5 ml were collected and measured using CLDB. The percentage of E protein in each fraction is displayed on the Y axis. doi:10.1371/journal.pone.0008325.g008 effective method to increase the expression level of glycoproteins from various viruses [35, 36] . Thus, gene optimization significantly enhanced expression of prME glycoproteins in transfected cells, and this over-expression increased both RSP production and secretion. However, it is also possible that using other viral strains may result in different efficacy of RSPs production, regardless of codon optimization. To our knowledge, this is the first report of RSPs using the native prM and E envelope proteins for the four serotypes of DVs in mammalian cells.