Membrane topology and processing of the minor glycoproteins of equine arteritis virus

Diese Dissertation enthält Journal-Beiträge, die in der gedruckten Version nicht enthalten sind. Die entsprechenden DOIs sind angegeben, so dass die Beiträge gegen Gebühr herunter geladen werden können. The minor envelope proteins of Arteriviruses gained more attention in recent years due to the discovery of the PRRSV receptor CD163 and subsequent studies indicating that this receptor interacts with Gp2 and Gp4 (Calvert et al., 2007; Das et al., 2010). As the minor proteins are dispensable for virus assembly and budding, but essential for virus infectivity, it was first assumed and then experimentally proven that they are responsible for cellular tropism of Arteriviruses (Tian et al., 2012). In spite of their important role in virus entry, there is little data available about arteriviral minor glycoproteins. The membrane topology, for instance, was only investigated in in vitro systems with canine microsomal membranes, but not in transfected or infected cells. In this thesis, I aimed to understand the processing of minor glycoproteins, with emphasis on Gp3. Previously published in vitro studies indicated that uncleaved SP of Gp3 may act as a membrane anchor alone, or that protein is anchored to membrane via N- and Ctermini. The aim was to experimentally confirm the topology model of Gp3, and address which part of the protein is responsible for its anchoring with the membrane. The other aim was to investigate the impact of the N-linked glycosylation adjacent to predicted SP cleavage site on the retention of SP in Gp3. Does the presence of the glycans just downstream cleavage site interfere with SP cleavage in Gp3? What are possible implications for EAV infectivity? The Gp2/Gp3/Gp4 trimeric complex is a likely candidate to perform receptor binding and fusion of Arteriviruses, but it is very difficult to study its function due to ER retention, formation of complex in ER and extracellular, small amounts of complex in the virions, lack of good antibodies, and limitations of reverse genetic system due to nested genome. Therefore, another aim of my work was to express the minor glycoprotein complex on the plasma membrane. Such expression would be a very promising tool for future functional studies and the role of the Gp2/Gp3/Gp4 trimer in Arteriviruses. Also, the oligomerisation between Gp2 and Gp4 was found to be crucial for virus infectivity (Wieringa et al., 2003a). As the cysteine responsible for the covalent bond between these two glycoproteins is only known for the Gp2, another aim was to investigate the partner cysteine in the Gp4.