Charles Weissmann - US grants

[unreadable] DESCRIPTION (provided by applicant): Transmissible spongiform encephalopathies, such as variant Creutzfeldt-Jakob disease in humans and "mad cow disease" in cattle are caused by an infectious agent called "prion". Prions consist mainly, if not exclusively, of PrPSc, a multimer consisting of misfolded form(s) of the ubiquitous host glycoprotein PrPC. Replication of PrPSc is believed to follow the "seeding model", whereby infecting PrPSc accrues host PrPC molecules that change their conformation as they add onto the PrPSc aggregates. Expression of PrP is essential but not sufficient for the propagation of prions; it is not known which other components are required. Prions affecting any one organism can occur in the form of many distinct strains which show quite different phenotypic properties, such as incubation time between infection and appearance of clinical disease, the location of lesions and sites of PrPSc accumulation in the brain. The fact that many different prion strains can be propagated indefinitely in the same inbred line of mice homozygous for the PrP gene is explained by the proposal that the strain is encoded by conformation of PrPSc. Prions can be propagated in vitro, but only in selected cell lines. In addition, cell lines are specific in their capacity to replicate some prion strains but not others, which may reflect the specificity of brain regions for this process. The aims of this project are to perfect the Scrapie Cell Assay (SCA), which allows the sensitive and rapid quantification of prions in cell culture rather than by titration in mice, to establish a panel of cell lines that allow prion strain identification by the SCA, to ascertain the fidelity of prion strain replication in cell culture and in cell-free systems by the Protein Misfolding Cyclic Amplification (PMCA). Using the tools we are developing, we intend to identify the components other than PrPC that are necessary to allow prion replication and strain recognition by cell lines (and ultimately tissues). The identification of components essential for prion replication will provide additional targets, beyond PrP, for prophylactic and therapeutic interventions against prion diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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- replicates 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.