Project Details
Projekt
The role of the GP5-M Spike of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) for virus budding and viral persistence
Subject Area
Veterinary Medical Science
Term
from 2010 to 2020
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 193516483
The porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped negative stranded RNA-virus in the Arteriviridae family, is the most important pathogen in the porcine industry. Glycoprotein 5 that forms a disulphide-link with the M protein is the main envelope protein, essential for virus budding and an important antibody target. PRRSV causes persistent infection, which is, besides the high variability of its glycoproteins, the main obstacle to elimination of the virus from pig populations. One hypotheses on the mechanistic basis for persistence, the decoy hypothesis, posits that evasion of adaptive immune responses during the initial phase of virus replication allows establishment of persistent infection. According to this model, antibodies against GP5 are made shortly after infection but do not neutralize the virus. Neutralizing antibodies appear only weeks into the infection to finally clear the virus. The epitopes of both types of antibodies on GP5 are located in close proximity to each other, separated by a hypervariable region with an equally variable number of carbohydrates. The binding site for the non-neutralizing antibodies was proposed to be a decoy epitope, against which antibodies are made that prevent the generation or binding of neutralizing antibodies. However, the decoy epitope is located in the signal peptide, a molecular domain required for targeting of GP5.In the last funding period we showed that the signal peptide is cleaved from GP5 of various PRRSV strains, independent of carbohydrates in its vicinity. Two signal peptide cleavage sites were identified for GP5 of genotype 2 PRRSV strains by mass spectrometry. Variable cleavage results in the production in two populations of GP5 proteins in virus particles, one without and the other with the decoy epitope. The first objective of his proposal is to generate recombinant PRRSV that contains GP5 cleaved only at site 1 or at site 2. The resulting virions then contain a homogenous population of GP5 molecules, either completely lacking or retaining the decoy epitope. In subsequent experiments recombinant viruses will be used to infect piglets to test the decoy hypothesis. The second objective is to functionally explore budding of PRRSV that includes GP5-M as its central element. It will be analyzed whether co-expression of GP5 and M induces the formation of VLPs, particles containing the viral proteins embedded in a membrane and having the same density and size as authentic virions. We will then investigate whether cytoplasmic tails of GP5-M interact with the N protein to recruit the viral genome to the budding site. We will determine whether membrane-proximal cysteines in GP5 and M are palmitoylated and whether this modification induces the formation of GP5-M oligomers as the driving force for virus budding. Finally, by expression of a dominant-negative mutant of VPS, we will determine if the cellular ESCRT module is required for release of virus particles.
DFG Programme
Research Grants
