Mutability of prions.

Murine prions transferred from brain to cultured cells gradually adapt to the new environment. Brain-derived 22L prions can infect neuroblastoma-derived PK1 cells in the presence of swainsonine (swa); that is, they are 'swa resistant'. PK1 cell-adapted 22L prions are swa sensitive; however, propagation in swa results in selection of swa-resistant substrains. Cloned, PK1 cell-adapted 22L prions were initially unable to develop swa resistance ('swa incompetent'); however, after serial propagation for 30-90 doublings, four of nine clones became swa competent, showing that swa-resistant 'mutants' arose during replication. Mutations in the case of prions are attributed to heritable changes in PrP(Sc) conformation. One clone remained swa incompetent even after 10(35)-fold expansion; surprisingly, after propagation in brain, it yielded swa-resistant prions, indistinguishable from the original 22L population. Thus, cell-adapted 22L prions assumed either mutable or virtually immutable conformations; however, when passaged through the brain all became mutable. Mutability is thus a substrain-specific attribute.

Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication.

Neuroblastoma-derived N2a-PK1 cells, fibroblastic LD9 cells, and CNS-derived CAD5 cells can be infected efficiently and persistently by various prion strains, as measured by the standard scrapie cell assay. Swainsonine, an inhibitor of Golgi α-mannosidase II that causes abnormal N-glycosylation, strongly inhibits infection of PK1 cells by RML, 79A and 22F, less so by 139A, and not at all by 22L prions, and it does not diminish propagation of any of these strains in LD9 or CAD5 cells. Misglycosylated PrP(C) formed in the presence of swainsonine is a good substrate for conversion to PrP(Sc), and misglycosylated PrP(Sc) is fully able to trigger infection and seed the protein misfolding cyclic amplification reaction. Distinct subclones of PK1 cells mediate swainsonine inhibition to very different degrees, implicating misglycosylation of one or more host proteins in the inhibitory process. The use of swainsonine and other glycosylation inhibitors described herein enhances the ability of the cell panel assay to differentiate between prion strains. Moreover, as shown elsewhere, the susceptibility of prions to inhibition by swainsonine in PK1 cells is a mutable trait.

Evaluation of removal of prion infectivity from red blood cells with prion reduction filters using a new rapid and highly sensitive cell culture-based infectivity assay.

BACKGROUND: The clearance of infectious prions from biologic fluids is usually quantified by bioassays based on intracerebral inoculation of hamsters or mice; these tests are slow, cumbersome, imprecise, and very expensive. In the present study we describe the use of a new and highly sensitive cell culture-based infectivity assay to evaluate the performance of several prion removal prototype filters. STUDY DESIGN AND METHODS: Five units of 1- to 2-day-old ABO-compatible human red blood cells (RBCs) in saline-adenine-glucose-mannitol were obtained from an AABB-accredited blood bank. The 5 units were combined to create a homogenous pool. Scrapie-infected mouse brain homogenate of a Rocky Mountain Laboratory strain was added to the pooled RBCs. The pooled RBCs were divided into 300-mL aliquots, which were filtered with either standard leukoreduction filter or four prototypes of prion reduction filter. The levels of prion infectivity in the pre- and postfiltration samples were measured with a cell culture-based standard scrapie cell assay (SSCA). RESULTS: All the 22-layer prion reduction filters removed prion infectivity below the limit of detection of the SSCA (reduction in prion infectivity > or =2.0 log(10)LD(50)/mL) while the 10-layer variant showed some residual infectivity. CONCLUSIONS: These results demonstrate the utility of a highly sensitive cell culture-based infectivity assay for screening prion reduction filters. The use of this type of in vitro infectivity assay will substantially help expedite the screening and discovery of devices aimed at reducing the risk of variant Creutzfeldt-Jakob disease transmission through blood transfusion.

Assaying prions in cell culture: the standard scrapie cell assay (SSCA) and the scrapie cell assay in end point format (SCEPA).

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.

Prion strain discrimination in cell culture: the cell panel assay.

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).