Generation of genuine prion infectivity by serial PMCA.
Prions are the causative infectious agents of transmissible spongiform encephalopathies (TSEs). They are thought to arise from misfolding and aggregation of the prion protein (PrP). In serial transmission protein misfolding cyclic amplification (sPMCA) experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalysed the structural conversion of cellular prion protein (PrP(C)) as efficiently as PrP(Sc) from the brain of scrapie-infected (263K) hamsters confirming an autocatalytic misfolding cascade as postulated by the prion hypothesis. However, the fact that PrPres generated in vitro was associated with approximately 10 times less infectivity than an equivalent quantity of brain-derived PrP(Sc) casts doubt on the "protein-only" hypothesis of prion propagation and backs theories that suggest there are additional molecular species of infectious PrP or other agent-associated factors. By combining sPMCA with prion delivery on suitable carrier particles we were able to resolve the apparent discrepancy between the amount of PrPres and infectivity which we were then able to relate to differences in the size distribution of PrP aggregates and consecutive differences in regard to biological clearance. These findings demonstrate that we have designed an experimental set-up yielding in vitro generated prions that are indistinguishable from prions isolated from scrapie-infected hamster brain in terms of proteinase K resistance, autocatalytic conversion activity, and - most notably - specific biological infectivity.
Weber
Petra
P
Centre for Neuropathology and Prion Research, Ludwig Maximilians University of Munich, Feodor-Lynen-Strasse 23, 81377 Munich, Germany. Petra.Weber@med.uni-muenchen.de
Giese
Armin
A
Piening
Niklas
N
Mitteregger
Gerda
G
Thomzig
Achim
A
Beekes
Michael
M
Kretzschmar
Hans A
HA
D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western / Q000662:veterinary; D002384:Catalysis; D006224:Cricetinae; D019286:Endopeptidase K / Q000378:metabolism; D008647:Mesocricetus; D018621:PrPC Proteins / Q000737:chemistry / Q000378:metabolism; D018620:PrPSc Proteins / Q000737:chemistry / Q000378:metabolism; D017096:Prion Diseases / Q000378:metabolism / Q000635:transmission / Q000662:veterinary*; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding
D018621:PrPC Proteins; D018620:PrPSc Proteins; D011328:Prions; D019286:Endopeptidase K