The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre- Golgi compartment

It was previously shown that transient transfection of RV cDNAs in COS cells resulted in adequate levels of expression for biochemical analysis of RV proteins (19) ; however, when the cells were examined by EM, numerous autophagic vacuoles and lysosomes were present most likely due to uptake of the DEAE-dextran used in the transfection procedure. To circumvent this and other possible cellular perturbations associated with transient transfection, we constructed stably transfected cells lines expressing moderate levels of E1 and E2 together or E1 alone. Stable cell lines were obtained by cotransfecting dhfr-CHO cells with plasmids containing genes for RV structural proteins and the plasmid pFR400 which contains the selectable marker dhfr (57) . Selection was based on the ability of transfectants to grow in nucleoside-free medium. Clones were isolated and screened for expression of RV antigens by indirect immunofluorescence and radioimmunoprecipitation. E1 glycoprotein in CHO ceils expressing both RV E2 and E1 (CHOE2E1 cells) was localized in the Golgi region (Fig. 1, A and B) , whereas in CHO cells expressing E1 alone (CHOE1 cells), the glycoprotein was localized in large compact structures often located adjacent to the nucleus ( Fig. 1 C) . In some ceils only a single large structure (up to 2-3 t~m) was observed, while in others, two or more larger staining masses were evident in addition to many smaller ones. These structures were not part of the Golgi complex since they were not stained by antibodies to o~-mannosidase II ( Fig. 1 D) . Incubating cells in cycloheximide for up to 6 h did not affect the distribution of E1 (not shown). CHO ceils constitutively expressing E2 alone also did not contain the heavily stained cytoplasmic structures found in CHOE1 cells. Instead E2 antigen was distributed throughout the RER and Golgi membranes and at the cell surface (not shown). The results indicated that when E2 and E1 are expressed together they are targeted to Golgi elements, whereas E1 accumulates in a compartment of unusual morphology when expressed without E2.