Fachbereich Veterinärmedizin



    Membrane topology and processing of the minor glycoproteins of equine arteritis virus (2014)

    Matczuk, Anna Karolina (WE 5)
    Berlin: Mensch und Buch Verlag, 2014 — 109 Seiten
    ISBN: 978-3-86387-473-5
    URL (Volltext): http://www.diss.fu-berlin.de/diss/receive/FUDISS_thesis_000000096791
    Institut für Virologie

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14163 Berlin
    +49 30 838 51833

    Abstract / Zusammenfassung

    Equine arteritis virus (EAV) is an enveloped RNA virus from the family Arteriviridae, which causes equine viral arteritis, mainly associated with abortions in mares. The trimer composed of the minor envelope glycoproteins Gp2, Gp3 and Gp4 is required for cell entry. These glycoproteins are predicted to be type I membrane protein with cleavable signal peptide (SP). To determine membrane topology in cells, the localisation of YFP fused C-terminally to the glycoproteins was analysed. In contrast to Gp2b and Gp4, the fluorescent tag of Gp3 localised in the lumen of the ER suggesting that the signal peptide (SP) functions as an uncleaved signal anchor (type II topology).
    The analysis of the glycosylation of Gp3 revealed that an overlapping sequon NNTT, adjacent to the SP cleavage site, was modified at both asparagines, although not in every Gp3 molecule. The presence of at least one glycosylation site in this region prevented SP cleavage in Gp3. Deletion of the overlapping sequon and blocking the glycosylation allowed SP cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable SP. The same phenomenon was observed in recombinant viruses, but surprisingly the infectivity of the EAV lacking the SP was not impaired. Membrane fractionation of transfected cells revealed that the uncleaved SP of the Gp3 was not responsible for membrane attachment of the protein. The membrane anchoring was achieved by the hydrophobic C-terminus, as its deletion allowed secretion of the protein to the supernatant. As a result of my study I propose a new topology model of Gp3, where the uncleaved SP is completely translocated into the ER lumen. The protein is anchored in the membrane via its hydrophobic C-terminus, which however does not span the membrane completely.
    Additionally, in order to express the Gp2/4 heterodimer on the plasma membrane for functional studies on this complex, the transmembrane regions and cytoplasmic tails were exchanged with corresponding regions of haemagglutinin of Influenza A. The proteins were able to form disulphide-linked Gp2/4 heterodimers, but were not targeted to the cell surface. In addition, single cysteines in the ectodomain of Gp4 were mutated, to determine the responsible one for the covalent linkage to Gp2. These mutations in transiently expressed Gp2/4, did not abolish dimer formation, but led to non-infectious virions in the viral context. The presented data about the minor glycoproteins of the EAV may facilitate studies on the Arterivirus entry. The SP retention mechanism observed in Gp3 could also apply for the other mammalian proteins. Future studies are needed to reveal the molecular basis of the unusual translocation and double sequon glycosylation of the Gp3.