Vaccinia-vectored expression of the rubella virus structural proteins and characterization of the E1 and E2 glycosidic linkages

Abstract

The maturation of rubella virus (RV) glycoprotein E2 from the single intracellular species (E2i; MW = 40 kDa) to the heterodisperse virion species (E2v; MW = 42 to 47 kDa), was studied by pulse-chase radiolabeling in Vero cells infected with RV or with recombinant vaccinia viruses (VVs) which express the entire RV structural protein open reading frame (VV-CE2E1) or glycoprotein E2 independently (VV-E2). The RV proteins expressed by the recombinant VVs comigrated with authentic RV intracellular proteins. In pulse-chase experiments, performed in both RV- and VV-CE2E1-infected cells, the amount of pulse-labeled E2i was substantially reduced during a 3- to 4-hr chase; during the same chase the amount of pulse-labeled E1 and C did not change. The concomitant appearance of the E2v forms was not observed. In contrast, in VV-E2-infected cells, no reduction in the amount of E2i occurred after as long as a 10-hr chase. Western blots using anti-E2 monoclonal antibodies showed that E2i was the predominant E2 species in cells infected with RV, VV-CE2E1, and VV-E2. However, minor amounts of three discrete species which comigrated within the extent of the E2v smear were also detected in cells infected with all three viruses, indicating that some degree of intracellular processing to E2v did occur. The disappearance of E2i during pulse-chase radiolabeling without the concomitant appearance of detectable E2v and the predominance of this labile form under steady-state conditions as revealed by Western blot analysis suggested that E2i was selectively turned over in both RV- and VV-CE2E1-infected cells. Such turnover was not apparent in VV-E2-infected cells, indicating that association with C and E1 was necessary for turnover to occur. Endoglycosidase digestion experiments and glycan differentiation assays revealed that E2v contained O-linked glycans. The presence of O-glycans on E2v accounted for part of the difference in size between E2v and E2i. Both virion E1 and E2 were found to contain high-mannose, hybrid-type, and complex-type N-glycans. Heterogeneity existed in the extent of processing of these glycans among individual E1 and E2 molecules.