Publikationsdatenbank

Membrane topology and processing of glycoprotein 3 (GP3) of porcine respiratory and reproductive syndrome virus (2018)

Art

Hochschulschrift

Autor

Zhang, Minze (WE 5)

Quelle

Berlin, 2018 — IX, 102 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/22990

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The porcine reproductive and respiratory syndrome virus (PRRSV) causes one of the most important infectious disease of pigs. PRRSV infects pigs of all ages, where it causes reproductive failure in sows and respiratory problems in piglets. Usually, symptoms are mild, but lead to reduced weight gain, which causes huge financial losses in the pork industry worldwide. In China even highly pathogenic strains emerged that kill 90% of infected pigs. So far, vaccines failed to eliminate the virus, which is due to the large variation between strains and their ability to escape the immunity of the host. The glycoprotein GP3 consists of an N-terminal signal peptide, a 180 amino acids long and highly glycosylated domain, a hydrophobic conserved region (20 aa) and a variable unglycosylated C-terminal domain (50-60 aa). GP3 is supposed to form a complex with two other glycoproteins (GP2 and GP4) in virus particles, but secretion of the protein from infected cells has also been reported. Here I analyzed the membrane topology of GP3 from type-1 and -2 PRRSV strains. First, Ifound that the N-terminal signal peptide of GP3 (and also from lactate dehydrogenase-elevating virus) is cleaved despite the presence of a carbohydrate in its vicinity. This is in contrast to GP3 of equine arteritis virus where a carbohydrate attached at a similar position prevents processing. Second, I confirmed that a fraction of wild-type GP3 is secreted from transfected cells; GP3 from PRRSV-1 strains (Lelystad, Lena) to a greater extent than GP3 from PRRSV-2 strains (VR-2332, IAF-Klop, XH-GD). This secretion behavior is reversed after exchange of the variable C-terminal domain. In contrast to intracellular GP3, secreted GP3 contains complex-type carbohydrates, indicating that it passed through the secretory pathway. Since intracellular and secreted GP3 have identical SDS-PAGE mobility after deglycosylation, the secreted form is not derived from proteolytic cleavage. Next I used a fluorescence protease protection assay to show that the C terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminal part of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region, but not of the variable C-terminus greatly enhances secretion of GP3. In addition, fusion of the hydrophobic region of GP3 to GFP promotes complete membrane anchorage of this (otherwise soluble) protein. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. This is consistent with the proposed role of GP3 in virus entry. In sum, GP3 exhibits a very unusual hairpin-like membrane topology. The signal peptide is cleaved and the C-terminus is exposed to the lumen of the ER. Membrane attachment is caused by a short hydrophobic region, which might form an amphiphilic helix. This rather weak membrane anchoring might explain why a fraction of the protein is secreted. We speculate that secreted GP3 might function as a “decoy”, which distracts antibodies away from virus particles.