NR AOXI
AU Nikles,D.; Merten,C.; Boller,K.; Kalinke,U.; Montrasio,F.; Cichutek,K.; Buchholz,C.J.
TI Displaying the prion protein on retrovirus-like particles
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - IV-22
PT Konferenz-Poster
AB
Retroviruses are enveloped viruses that can form particles and bud from the cell membrane of infected cells in the absence of their envelope protein. Previous work from our group and others showed that foreign polypeptides can be displayed on the surface of retroviral particles. This can be achieved either by random incorporation of overexpressed transmembrane proteins or by displaying polypeptides on top of the trimeric envelope protein (Env).
To set up a retroviral display system for the murine prion protein molecule either the full length polypeptide or the C-terminal domain covering amino acids 121-231 were fused to the transmembrane domain of the platelet derived growth factor receptor (PDGFR) or to the N-terminus of the murine leukaemia virus (MLV) Env protein. The constructs were transfected into cells expressing the MLV or the HIV gag/pol genes coding for the viral capsid proteins. Western blot analysis of particles sedimented from the supernatant of the transfected cells revealed that the PrP-PDGFR protein as well as the PrP110-PDGFR and the PrP110-Env proteins had been successfully incorporated and showed the typical PrP glycosylation pattern. Particle numbers and incorporation efficiencies were highest with the PrP110-PDGFR construct, allowing particle numbers of above 10exp11/ml in concentrated stocks. Immunogold electron microscopy of transfected cells using the anti-PrP monoclonal antibody 6H4 showed a high density of gold particles on the outside of the cell membrane. In particle preparations PrP specific gold labeling was detected on the membranes of particles with a typical retroviral shape but also on pleomorphic vesicle-like structures.
The data demonstrate that the PrP molecule can be successfully displayed on retroviral particles. The described PrP-retroparticles constitute a novel tool to i) investigate the oligomerisation state of PrP molecules and ii) develop a vaccination strategy against TSEs.
AD D. Nikles, C. Merten, K. Boller, U. Kalinke, F. Montrasio, K. Cichutek, C.J. Buchholz, Paul-Ehrlich-Institut, 63225 Langen, Germany
SP englisch
PO Deutschland