Cell lysate (CL) and clarified supernatant (SN) concentrated by ultra-centrifugation were analyzed by Western blotting followed by incubation with either the anti-E monoclonal antibody 4E11 ( Figure 4A -B) or an anti-DV1 serum from a human patient ( Figure 4C -D). In CL samples, a 50 kDa monomeric form of the E glycoprotein was predominantly observed ( Figure 4A ,C), whereas high levels of 100 kDa E glycoprotein homodimers were readily detected in SN samples ( Figure 4A -D). This suggests that whereas in HeLa-prME cells the majority of viral proteins are localized in the early pre-Golgi secretory pathway in an immature state, secreted RSPs have passed through maturation stages in the Golgi apparatus thus resulting in homodimerization of E proteins. To further confirm the presence of E dimers in SN and exclude the possibility that dimerization resulted from treatment for electrophoresis, the SN were incubated in the presence or absence of a cross linker (3, 39-dithiobis [sulfosuccinimidylpropionate]; DTSSP) and then subjected to Western blotting using the anti-E 4E11 antibody. Under these conditions, we observed that E dimer/ monomer ratio was lower in non-treated RSPs compared to the Figure 3 . Electron microscopy analysis of the HeLa-prME cell line. HeLa-prME (DV1) cells were fixed and either prepared for epon embedding (A), or for immuno labeling on thawed cryosections (B-D). A), Round particles are found aligned in the lumen of the ER (arrows) together with tubular structures (arrowheads). B) Labeling for the E protein is present in the lumen of cisternae. It is localized to electron lucent, round particles (arrows) and tubular structures (arrowheads). In addition E protein is also found on amorphous, electron dense material present in these cisternae (asterix). C) Double labeling of E protein (10 nm gold, black arrows) and the ER marker calreticulin (15 nm gold, white arrows). Calreticulin is present on the limiting membrane of the cisternae, which contain round particles positive for E protein labeling. D) Label for the E protein is also found within the Golgi stack (G) and on vesicles in the Golgi area. N = nucleus, all scale bars represent 200 nm. doi:10.1371/journal.pone.0008325.g003 DTSSP treated (data not shown). These data indicate that, in SN, a significant proportion of E protein is present as a homodimer and detection of E monomers most likely results from partial dissociation under the denaturing conditions of electrophoresis. E homodimers were no longer detected when samples were heated in presence of dithiothreitol ( Figure 4B ). In addition, the prM protein could be readily detected with the anti-DV1 serum in CL but not SN samples ( Figure 4C ). By contrast, when the HeLa-prME cells were treated with NH 4 Cl, which inhibits acidification of the trans-Golgi compartment and, hence, the activity of furin protease and prM cleavage, prM was also found in SN ( Figure 4D -E). To our surprise E dimers were also detected in these conditions. Interestingly, the E monomers from SN exhibited slower electrophoretic mobility than those of CL samples, which suggests that E glycoproteins have acquired complex N-glycans in the Golgi apparatus. This was confirmed by N-Glycosidase F (PNGase F) and endoglycosidase H (EndoH) digestion ( Figure 4 F-G), which cleaves nearly all types of N-glycan chains or only high mannose and some hybrid N-glycan chains from glycoproteins, respectively. Whereas E protein in CL was sensitive to both treatments, only PNGase F digestion increased the electrophoretic mobility of E protein in SN, demonstrating that RSPs had acquired EndoH-resistant, complex N-glycans in the Golgi apparatus before secretion. Moreover, we found that secretion of DV RSPs was altered by incubation of cells at 15uC and 20uC, which block cargo transport from ERGIC to cis-Golgi and exit from the trans-Golgi, respectively, and by brefeldin A treatment, which inhibits exit from ER and induces fusion of Golgi membranes with ER (data not shown). Altogether, homodimerization of E, acquisition of complex sugars and efficient cleavage of prM indicate that prM and E viral proteins have correctly assembled in the ER into RSPs which have trafficked through the secretory pathway before secretion into the cell medium.