AbstractsBiology & Animal Science

Expression and characterization of spike protein complexes Gp2/Gp3/Gp4 and Gp5/M of the Arterivirus

by Sekhar Sinhadri Balaji Chandra

Institution: Freie Universität Berlin
Department: FB Biologie, Chemie, Pharmazie
Degree: PhD
Year: 2015
Record ID: 1103605
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000099365


Equine arteritis virus (EAV) and Porcine reproductive and respiratory syndrome virus (PRRSV) are enveloped RNA viruses belonging to the family Arteriviridae. EAV infection leads to abortion and respiratory illness in horses. PRRSV causes persistent infections in pigs, which is one of the main reasons for the economic losses in the swine industry. The glycoprotein complex Gp2/3/4 and Gp5/M are essential for cell entry and budding, respectively. PRRSV and EAV Gp2/3/4 ectodomains were co-expressed to determine their complex formation in insect cells. The results revealed that PRRSV Gp2/3/4 proteins secreted into the cell culture supernatant as a disulphide linked complex. In contrast, Gp3 of EAV was expressed, but not associated with Gp2/4. The Gp2/3/4 proteins of both PRRSV and EAV are attached to the membrane through their C-terminal hydrophobic transmembrane region as they were secreted when it is removed. This result showed that all the proteins contain type I membrane topology except EAV Gp3 since the C-terminus does not span the membrane. Mass spectrometry results demonstrated the exact signal peptide cleavage sites for Gp2, Gp4, and Gp3 of both PRRSV and EAV. The analysis of glycosylation showed that all predicted N- linked glycosylation sites are used in both EAV and PRRSV Gp2/3/4. EAV Gp3 possesses an uncleavable signal peptide. But, my results showed that the signal peptide is cleaved off from the insect cell expressed Gp3. The difference in size between mammalian and insect cell expressed EAV Gp3 (deglycosylated) indicated that the signal peptide cleavage is due to an unknown Gp3 specific processing difference in insect cells. EAV Gp2/4 proteins were able to form the disulphide linked complex but exist in aberrant multimeric forms. My results showed that cysteine residues in the Gp4 ectodomain have no influence on multimer formation, and any cysteine residue in the Gp4 ectodomain can compensate the disulphide linkage with Gp2. Mass spectrometry result of PRRSV Gp5 demonstrated that the signal peptide is cleaved at two sites, which are also predicted by the bioinformatics software. This finding indicates that the majority of the Gp5 molecules lack the decoy epitope, which is believed to mask the immune response from its neutralising epitope. Mutating the only cysteine residue in the Gp5 ectodomain after the SP cleavage demonstrated that it was responsible for the disulphide linkage between Gp5/M. The data produced in this study on minor and major glycoprotein complexes of EAV and PRRSV may be useful for future structural and functional studies to understand their role in cell entry. Das equine Arteritisvirus (EAV) sowie das porzine reproduktive und respiratorische Syndrom Virus (PRRSV) sind umhüllte RNA Viren, die zu der Familie der Arterivi-ren gehören. Während eine Infektion mit EAV in Pferden zu Fehlgeburten und respi-ratorischen Symptomen führen kann, kommt es bei PRRSV zu persistenten Infektio-nen von Schweinen und somit zu einem der größten ökonomischen Verluste in…