Structure and function of Gp3 of porcine reproductive and respiratory syndrome virus

点击翻译按钮获取中文摘要

The porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped negative stranded RNA-virus of the family Arteriviridae, is the most important pathogen in the porcine industry. Its membrane contains two glycoprotein spikes, Gp5/M, which is required for virus budding and a heterotrimeric Gp2/3/4 complex, which has been proposed to play a role during virus entry. However, our information about Gp2/3/4 is mainly derived from studies with the equine arteritis virus, the prototype member of the family. Very little is known about the respective proteins of PRRSV, which also exhibit large amino acid variability. We previously studied basic biochemical features of Gp3 from various PRRSV strains. A fraction of the protein is secreted both from infected and transfected cells, Gp3 from PRRSV-1 strains to a greater extent than Gp3 from PRRSV-2 strains. This secretion behaviour is reversed after exchange of the (between strains) variable C-terminal domain. We also showed that the protein exhibits an unusual hairpin-like membrane topology: both the N- and C-terminus are oriented towards the lumen of the endoplasmic reticulum (ER), membrane anchoring might occur via an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents secretion of Gp3 and in its hydrophobic face enhances it. This rather weak membrane anchoring explains why a fraction of the protein is secreted from cells. With this project we want to test the hypothesis that the hydrophobic region of Gp3 indeed forms an amphiphilic helix, as predicted by bioinformatics. We attempt to determine the structure of this region in the absence and presence of various artificial membranes using CD- and NMR-spectroscopy. Using reverse genetics we will investigate whether virus replication in cell culture requires that a fraction of Gp3 is secreted and whether the amino acid sequence of the hydrophobic region is essential, since it is highly conserved between PRRSV strains although amphiphilic helices can be formed by very different peptides. Next, we want to determine whether soluble and/or membrane-bound Gp3 associates with Gp2/4 and how the complex assembles. Finally, using purified ectodomains of Gp2/3/4 which assemble into disulfide-linked dimers and trimers, we will identify the cysteines that connect the proteins by disulfide-linkages and investigate with affinity chromatography whether Gp2/3/4 binds to CD 163, the essential receptor for cell entry of PRRSV.