The endoplasmic reticulum is the assembly site for flaviviruses. In order to verify whether this ER staining reflects the presence of E-containing assembled particles, we analyzed the HeLa-prME stable cell line by transmission electron microscopy (TEM) on thin sections of fixed, epon-embedded cells ( Figure 3A ). We found that HeLa-prME, but not control HeLa cells (data not shown), displayed dilated ER membranes with aligned round particles and elongated parallel tubules. Particles were ,20 nm in diameter and homogenous in size and shape. The aligned particles that we have observed in HeLa-prME cells are similar to the stacked viral particles which have been described in cisternae of the rough ER in infected insect and mammalian cells [25, 26, 27, 28] . To confirm that these structures contain viral proteins, we labeled thawed cryosections with the 4G2 antibody that recognizes the E protein ( Figure 3B -D). We found antibody binding for the E protein Figure 2 . Subcellular localization of the DV1 E protein in HeLa-prME cells. HeLa-prME cells were fixed, permeabilized, and stained for E protein (green) and for cellular marker antigens (red). DAPI staining was used to label cell nuclei (blue). Erp72, ERGIC-53 and Golgin-97 are proteins of the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment (ERGIC) and Golgi apparatus, respectively. The scale bar represents 10 mm. doi:10.1371/journal.pone.0008325.g002 present in the lumen of cisternae, where it is localized to electron lucent, round particles and tubular structures. In addition, E protein is also found on amorphous, electron dense material present in these cisternae ( Figure 3B ). To confirm the nature of these cisternae by other than morphological criteria we performed a double labeling of the 4G2 antibody with the ER resident protein calreticulin. We found antibody binding for calreticulin present on the 4G2 positive cisternae ( Figure 3C ), unequivocally identifying them as ER. In addition, we found a lower but significant amount of label for the E protein present in the Golgi stack and in vesicles in the Golgi area ( Figure 3D ).