Prions are infectious proteins consisting mainly of PrP Sc , a β sheet-rich conformer of the normal host protein PrP C , and occur in different strains. Strain identity is thought to be encoded by PrP Sc conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating "mutants", and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, "cell-adapted" prions outcompeted their "brain-adapted" counterparts, and the opposite occurred when prions were returned from cells to brain. Similarly, the inhibitor swainsonine selected for a resistant substrain, while in its absence the susceptible substrain outgrew its resistant counterpart. Prions, albeit devoid of a nucleic acid genome, are thus subject to mutation and selective amplification.

Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).standard scrapie cell assay ͉ infectivity ͉ response index ͉ PrP

Sc, an aggregated conformer of the host protein PrP C . Prions come in different strains, all based on the same PrP C sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected.

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.

Human prion diseases have inherited, sporadic, and acquired etiologies. The appearance of the novel acquired prion disease, variant Creutzfeldt-Jakob disease (vCJD), and the demonstration that it is caused by the same prion strain as that causing bovine spongiform encephalopathy, has led to fears of a major human epidemic. The etiology of classical (sporadic) CJD, which has a worldwide incidence, remains obscure. A common human prion-protein-gene (PRNP) polymorphism (encoding either methionine or valine at codon 129) is a strong susceptibility factor for sporadic and acquired prion disease. However, a quantitative-trait-locus study of prion incubation periods in mice has demonstrated an important factor that is close to Prnp but is independent of its coding sequence or that of the nearby prion-like doppel gene (Prnd). We have analyzed the PRNP locus for such tightly linked susceptibility factors. Fifty-six polymorphic sites have been identified within 25 kb of the PRNP open reading frame, including sites within the PRNP promoter and the PRNP 3' untranslated region. These have been characterized in 61 Centre d'Etude du Polymorphisme Humain (CEPH) families, demonstrating extensive linkage disequilibrium around PRNP and the existence of 11 major European PRNP haplotypes. Haplotype frequencies estimated in healthy U.K. control individuals were very similar to those deduced in the CEPH families. A common haplotype was overrepresented in patients with sporadic CJD (sCJD). Through use of a log-linear modeling approach to simultaneously model Hardy-Weinberg and linkage disequilibria, a significant independent association was found between sCJD and a polymorphism upstream of PRNP exon 1 (P=.005), in addition to the strong susceptibility conferred by codon 129 (P=2x10(-8)). However, although our sample size was necessarily small, no association was found between these polymorphisms and vCJD or iatrogenic CJD, in keeping with their having distinct disease mechanisms. In addition, there was no evidence of a PRNP founder effect in the first reported geographical cluster of vCJD.

Prions are usually quantified by bioassays based on intracerebral inoculation of animals, which are slow, imprecise, and costly. We have developed a cell-based prion assay that is based on the isolation of cell lines highly susceptible to certain strains (Rocky Mountain Laboratory and 22L) of mouse prions and a method for identifying individual, prion-infected cells and quantifying them. In the standard scrapie cell assay (SSCA), susceptible cells are exposed to prion-containing samples for 4 days, grown to confluence, passaged two or three times, and the proportion of rPrP(Sc)-containing cells is determined with automated counting equipment. The dose response is dynamic over 2 logs of prion concentrations. The SSCA has a standard error of +/-20-30%, is as sensitive as the mouse bioassay, 10 times faster, at least 2 orders of magnitude less expensive, and it is suitable for robotization. Assays performed in a more time-consuming end point titration format extend the sensitivity and show that infectivity titers measured in tissue culture and in the mouse are similar.

Prions consist mainly of PrP Sc , a pathogenic conformer of hostencoded PrP C . Prion populations with distinct phenotypes but associated with PrP Sc , having the same amino acid sequence, constitute distinct strains. Strain identity is thought to be encoded by the conformation of PrP Sc and to be maintained by seeded conversion. Prion strains can be distinguished by the cell panel assay, which measures their ability to infect distinct cell lines. Brainderived 22L prions characteristically are able to infect R33 cells (i.e., are "R33 competent"), as well as PK1 cells in the presence of the inhibitor swainsonine (i.e. are "swa resistant"). Here we report that 22L prions retained their characteristic cell tropism and swa resistance when transferred from brain to R33 cells. However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. PrP Sc associated with swaresistant/R33-competent and swa-sensitive/R33-incompetent prions had different conformational stabilities. When cloned R33-incompetent/swa-sensitive prions were again propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions. Our results support the view that 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments.evolution | scrapie cell assay | selection W e determine the susceptibility of a cell line to a prion strain by the standard scrapie cell assay (SSCA) (1, 2). In short, we expose cells to various dilutions of the prion sample, propagate them for three splits, and determine the proportion of PrP Sc -containing cells by ELISA. We define as response index (RI) the reciprocal of the dilution that yields a designated proportion of infected cells under standard conditions (usually 300 PrP Sc -positive cells/20,000 cells).We discriminate murine prion strains by the cell panel assay (CPA) (3), which is based on the differential susceptibility of selected cell lines to individual prion strains. RML, 22L, ME7, and 301C prions can be distinguished by their relative RI values on a panel consisting of neuroblastoma-derived PK1 cells in the presence or absence of swainsonine (swa), neuroblastoma-derived R33 (or, recently, the more susceptible subclone R33 2H11 ) cells, fibroblastic LD9 and CNS-derived CAD cells. Swa, an inhibitor of complex glycosylation (4), suppresses infection of PK1 cells by RML but not by 22L prions (5).As recently reported, when 22L prions were transferred from brain to cultured PK1 cells, the PK1 cell-adapted 22L variants gradually outgrew the original population, as documented by a profound change in their CPA characteristics: Although brainderived 22L prions efficiently infected R33 cells (i.e., were R33 competent) and PK1 cells in the presence of swa (i.e., were swa resistant), the PK1 cell-adapted 22L prions were R33 incompetent (i.e., were 1.5-2 logs less infec...

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP Sc , an abnormal conformer of the host glycoprotein PrP C . TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP Sc with the same amino-acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post-translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild-type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N-glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild-type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain-specific characteristics of the 79A TSE strain changed when PrP Sc was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post-translational changes to PrP which we propose result in the selection of mutant TSE strains.