Processing and intracellular transport of rubella virus structural proteins in COS cells

Abstract

Plasmids encoding rubella virus (RV) structural proteins C-E2-E1, E2-E1, E2, and E1 have been constructed in the eukaryotic expression vector pCMV5. The processing and intracellular transport of these proteins have been examined by transient expression of the cDNAs in COS cells. Compared to alphaviruses, processing of RV glycoprotein moieties occurred relatively slowly and the transport of glycoproteins E2 and E1 to the plasma membrane was inefficient. Indirect immunofluorescence revealed that the majority of RV antigen in transfected and infected COS cells was localized to the Golgi region, including the capsid protein. Accumulation of capsid protein in the juxtanuclear region was determined to be RV glycoprotein dependent. Unlike alphaviruses, RV E1 did not require E2 for targeting to the Golgi where it was retained. E2 was however necessary for cell surface expression of E1. This study revealed that the processing and transport of RV structural proteins is quite different from alphaviruses and that the accumulation of antigens in the Golgi region may be significant in light of previous reports which suggest that RV buds from the internal membranes in some cell types.

Intracellular transport of rubella virus structural proteins expressed from cloned cDNA

“…Immunofluorescence studies revealed that the mutant C protein was present in the cells as discrete bodies and clusters of bodies; the C_s~ protein appears to dimerize as normal, and it is possible that it is also forming larger aggregates, or associating with other, as yet unidentified subcellular structures. GiUam and coworkers (Hobman et al, 1990;McDonald et al, 1991) have reported that the normal C is distributed throughout the cytoplasm, with or without the E2 protein. It is difficult to explain these findings in the light of both our own immunofluoresence studies and the results of our in vitro and in vivo membrane association assays.…”

“…These biochemical data are confirmed, to some extent, by immunofluorescence studies of the location of the RV structural proteins when expressed in various combina- tions. These suggest that E2 and E1 co-localize in a Golgi-like compartment in COS cells when expressed together, with or without the C protein (Hobman et al, 1990; our own unpublished observations). In addition, E2, when expressed alone, was found distributed in both the ER and the Golgi complex (Hobman et al, 1990; our own unpublished observations).…”

“…These suggest that E2 and E1 co-localize in a Golgi-like compartment in COS cells when expressed together, with or without the C protein (Hobman et al, 1990; our own unpublished observations). In addition, E2, when expressed alone, was found distributed in both the ER and the Golgi complex (Hobman et al, 1990; our own unpublished observations). However, the absence of Golgi-specific processing of E1 if this protein is expressed without E2 is at odds with the reported location of E 1 in the Golgi in such cells (Hobman et al, 1990).…”

“…In addition, E2, when expressed alone, was found distributed in both the ER and the Golgi complex (Hobman et al, 1990; our own unpublished observations). However, the absence of Golgi-specific processing of E1 if this protein is expressed without E2 is at odds with the reported location of E 1 in the Golgi in such cells (Hobman et al, 1990). In our immunofluorescence studies we observed El, expressed on its own, in large vesicular structures either distributed in the cytoplasm (Fig.…”

“…It is therefore of considerable interest to investigate the synthesis and transport of these proteins in isolation from virion formation, to determine whether or not they reach the plasma membrane and, if not, to determine by what mechanism intracellular retention is achieved. In a recent study (Hobman et al, 1990), immunofluorescence was used to show that E2 was transported to the surface of COS cells when expressed alone or with E 1, but that E1 required the presence of E2 for surface expression. This study, however, did not directly examine the proportion of E2 and E 1 located at the cell surface.…”