Prions, the pathogens causing transmissible spongiform encephalopathies, are unique in that they consist mainly, if not entirely of multimers of PrPSc, a conformational isomer of the host- encoded protein PrPC. Prions occur in the form of diverse strains, which differ in various phenotypic properties but whose PrPSc has the same amino acid sequence. It is believed that strain identity is encoded by the conformation of PrPSc and that there are as many distinct conformations as there are strains. We have observed that a prion strain, 22L, when transferred from brain to cell culture, changed its characteristics, but gradually regained them when re-introduced into brain. Moreover, when propagated in cell culture in the presence of an inhibitory drug, swainsonine, the prion population acquired resistance against the drug, but lost the resistance when propagated for prolonged periods in its absence. This is quite remarkable, considering that bacteria and viruses acquire drug resistance as a consequence of random mutations in their genetic material, whereas prions do not contain informational nucleic acids. Our working hypothesis is that a prion strain population is comprised not of PrPSc with a unique conformation but of a multitude of species ("sub-strains") with related conformations. This is analogous to the concept of "quasispecies" we first established for RNA phages three decades ago. We postulate that the replication rates of sub-strains may vary depending on the host cell. In a particular environment one of these species may represent the major component and out- replicate other conformers, but at the same time, variant conformers are continuously generated at some low rate (because the activation energy barriers for interconversion are low). Upon transfer to a different host or a change in the environment a different conformer may become the major constituent of the population. We wish to determine whether PrPSc associated with drug resistant and drug sensitive prions show physico-chemical differences that may reflect different conformations, and whether the two types of prions replicate at different rates in host cells. We will investigate whether prions can develop resistance against other inhibitory drugs. Importantly, we will determine whether prion populations that have never been exposed to swainsonine already comprise resistant variants, as would be expected from the "quasispecies" hypothesis. We intend to biologically clone a drug-sensitive prion and determine whether swainsonine-resistant variants arise during propagation in the absence of the drug, and if so, at what rate. Finally, we will ascertain whether it is possible to select prion "sub-strains" that are more resistant to chaotropic agents or to temperature, which would be further evidence in favor of heterogeneity of prion populations. The major technical innovation making this investigation possible is the Cell Panel Assay (CPA), which is based on the Standard Scrapie Cell Assay (SSCA), both of which were developed in our laboratories and which allow the rapid discrimination between prion strains. The finding that prions can acquire drug resistance impacts on the strategies envisaged for therapeutic approaches to prion disease.

Public Health Relevance

Prions are pathogens that consist of a misfolded version of a naturally occurring host protein and multiply within cells. We have found a drug that prevents prion multiplication in cell culture; surprisingly, exposure of prion-infected cells to this drug can result in the emergence of drug- resistant prions, the first example of its kind. This project is to investigate how drug-resistant prions arise, a question that has important consequences for the design of treatment strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS067214-03
Application #
8259850
Study Section
Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
Program Officer
Wong, May
Project Start
2010-05-15
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$424,463
Indirect Cost
$210,088
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Oelschlegel, Anja M; Weissmann, Charles (2013) Acquisition of drug resistance and dependence by prions. PLoS Pathog 9:e1003158
Cancellotti, Enrico; Mahal, Sukhvir P; Somerville, Robert et al. (2013) Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties. EMBO J 32:756-69
Wang, Fei; Zhang, Zhihong; Wang, Xinhe et al. (2012) Genetic informational RNA is not required for recombinant prion infectivity. J Virol 86:1874-6
Weissmann, Charles (2012) Mutation and selection of prions. PLoS Pathog 8:e1002582
Oelschlegel, Anja M; Fallahi, Mohammad; Ortiz-Umpierre, Shannon et al. (2012) The extended cell panel assay characterizes the relationship of prion strains RML, 79A, and 139A and reveals conversion of 139A to 79A-like prions in cell culture. J Virol 86:5297-303
Browning, Shawn; Baker, Christopher A; Smith, Emery et al. (2011) Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication. J Biol Chem 286:40962-73
Weissmann, Charles; Li, Jiali; Mahal, Sukhvir P et al. (2011) Prions on the move. EMBO Rep 12:1109-17
Li, Jiali; Mahal, Sukhvir P; Demczyk, Cheryl A et al. (2011) Mutability of prions. EMBO Rep 12:1243-50