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Molecular biological studies of rubella virus structural proteins Qiu, Zhiyong


Rubella virus (RV) is a small enveloped RNA virus in the Togaviridae family. The virion contains three structural proteins, a capsid protein (C) associated with the genomic RNA to form the nucleocapsid and two membrane glycoproteins, E1 and E2. The RV structural proteins are translated as a polyprotein precursor p110 (NH2-C-E2-E1-COOH) from a RV-speciflc 24S subgenomic RNA and derived by posttranslational processing of p110. The role of N-linked glycosylation of El and E2 on their respective biological functions has been studied by expressing glycosylation mutants of E1 and E2 generated by oligonucleotide-directed mutagenesis on coding cDNA. Expression of the E2 mutant proteins in COS cells indicated that removal of any of the glycosylation sites resulted in slower glycan processing, lower protein stability and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number and location of deleted carbohydrate sites. Expressed E1 glycosylation mutant proteins from vaccinia recombinants were recognized by a panel of E1-specific monoclonal antibodies, indicating that carbohydrate side chains on E1 are not involved in the constitution of epitopes recognized by these monoclonal antibodies. All the E1 glycosylation mutants were capable of eliciting anti-RV E1 antibodies in immunized mice; however, only the single glycosylation mutants were found to be capable of inducing viral neutralizing antibodies, suggesting that carbohydrates on E1 are important for maintaining proper protein folding and epitope exposure. Assembly of RV was found to be independent of the genomic RNA but strictly dependent upon the co-expression of C, E2 and E1, in stable cell lines expressing RV structural proteins. Assembly and release of RV virion was dramatically reduced in RV-infected cells treated with two Golgi transport inhibitors, brefeldin A and monensin, although there was no significant alteration for the expression and processing of the structural proteins. My finding indicates that stable association of RV E1 and E2 with the intact Golgi complex is essential for efficient RV assembly.

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