Recombinant Rubella Virus E2 protein

Very little is known about the cellular factors that are required for the maturation of rubella virus glycoproteins (E2 and E1) in the endoplasmic reticulum of the infected cell. In the present study, we established the interaction of the ER chaperone proteins, calreticulin and calnexin. with the RV El and E2 proteins in cells stably expressing the viral proteins. The interaction between E2 and calnexin was significantly higher than with calreticulin. In pulse-chase experiments, the half-life of the E2-calnexin was >45 min, whereas the half-life of the calreticulin-E2 interaction was similar to 10 min. Tunicamycin and castanosper mine treatments altered the mobilities of intracellular El and E2, due to either lack of oligosaccharide ligand addition or trimming of terminal glucose residues, respectively. Further, the drug treatments resulted in a loss of El and E2 interaction with calreticulin or calnexin, thereby demonstrating that the interaction is through monoglucosylated forms of RV proteins. These studies suggest that the interaction of RV glycoproteins with the ER chaperone proteins is essential for their maturation in the endoplasmic reticulum.

We have constructed a recombinant baculovirus expressing the rubella virus E2 (42-45 KDa) and C (34 KDa) proteins. Sf9 cells infected with recombinant virus were able to synthesize and process the two proteins coded by a unique precursor gene. By immunoblot and immunoprecipitation analysis with polyclonal and monoclonal antibodies, a precursor polyprotein (66 KDa) and two other proteins migrating with an apparent molecular weight of 42 KDa and 36KDa were recognized as E2 glycoprotein and C protein, respectively. The recombinant E2 protein appeared to be glycosylated since it was susceptible to tunicamycin. The results indicate that the RV polyprotein coding for E2 and C is expressed and proteolytically cleaved in insect cells. This baculovirus expression system provides a useful alternative approach for the production of rubella virus antigens and should allow the purification of large quantities of the RV proteins for further biochemical and immunological studies.