Recombinant RuV E1 Mosaic [His]

Rubella virus (RV) envelope glycoproteins, E2 and E1, form a heterodimeric complex that is targeted to medial/trans-Golgi cisternae. To identify the Golgi targeting signal(s) for the E2/E1 spike complex, we constructed chimeric proteins consisting of domains from RV glycoproteins and vesicular stomatitis virus (VSV)G protein. The location of the chimeric proteins in stably transfected Chinese hamster ovary cells was determined by immunofluorescence, immunoelectron microscopy, and by the extent of processing of their N-linked glycans. A trans-dominant Golgi retention signal was identified within the C-terminal region of E2. When the transmembrane (TM) and cytoplasmic (CT) domains of VSV G were replaced with those of RV E2, the hybrid protein (G-E2(TMCT+)) was retained in the Golgi. Transport of G-E2(TMCT+) to the Golgi was rapid (t(1/2)=10-20 min). The G-E2(TMCT+) protein was determined to be distal to or within the medial Golgi based on acquisition of endo H resistance but proximal to the trans-Golgi network since it lacked sialic acid. Deletion analysis revealed that only the TM domain of E2 was required for Golgi targeting. Although the cytoplasmic domain of E2 was not necessary for Golgi retention, it was required for efficient transport of VSV G-RV chimeras out of the endoplasmic reticulum. When assayed in sucrose velocity sedimentations gradients, the Golgi-retained G-E2(TMCT+) protein behaved as a dimer. Unlike virtually all other Golgi targeting signals, the E2 TM domain does not contain any polar amino acids. The TM and CT domains of El were not required for targeting of E2 and El to the Golgi indicating that a heterodimer of two integral membrane proteins can be retained in the Golgi by a single retention sigher.

Haemagglutination (HA) by rubella virus is mediated by the E1 glycoprotein. Rubella isolates which haemagglutinate with different avidity have been characterised. A significant reduction of HA titre at pH6.0 was observed in one isolate in which isoleucine is substituted for threonine at rubella E1 residue 280. This residue is located in an epitope (EP1) which we have previously identified and shown to bind HA inhibiting (HA1) monoclonal antibodies. The isolates studied are also distinguishable by plaque size but no sequence variations in the immunogenic region of E1 were identified which might account for this difference. No correlation was observed between infectivity and binding affinity of neutralising monoclonal antibodies for different rubella virus strains.