Molecular properties, partial purification, and assay by incubation period measurements of the hamster scrapie agent

Abstract

The scrapie agent causes a progressive degeneration of the CNS of animals after a prolonged incubation period. Measurements of incubation period length, defined as the time from inoculation to the onset of clinical signs of neurological dysfunction, were related to the titer of the agent and the dilution of the inoculated sample. Equations defining the relationship provide a new assay for the agent requiring fewer animals than end point titrations. By use of this incubation period assay, the scrapie agent from hamster brain had an s20,w of < 300 S but > 30 S assuming .rho.p = 1.2 g/cm3. A partially purified fraction P3 was obtained by differential centrifugation and sodium deoxycholate extraction. When P3 was extracted with phenol, virtually no infectivity was found in the aqueous phase even after examining such variables as pH, salt concentration and predigestion of samples with proteinase K. Nonionic and nondenaturing, anionic detergents did not inactivate the scrapie agent; denaturing detergents inactivated the agent. Sodium dodecyl sulfate [SDS] inactivated greater than 90% of the agent at a SDS protein ratio of 1.8 g/g. Inactivation by SDS appears to be a cooperative process. Addition of a nonionic detergent to form mixed micelles with SDS prevented inactivation of the agent by SDS. Weak chaotropic ions do not inactivate the scrapie agent, while strong chaotropic ions like SCN- and Cl3CCOO- destroy infectivity at concentrations of 0.2 M. These data provide evidence in support of a protein component within the scrapie agent which is essential for maintenance of infectivity. It is unlikely that the scrapie agent is composed only of a naked nucleic acid, as is the case for the plant viroids.