Scrapie prion proteins accumulate in the cytoplasm of persistently infected cultured cells

The cellular prion protein (PrP^c) is a sialoglycoprotein anchored to the external surface of cells by a glycosyl phosphatidylinositol moiety. During scrapie, an abnormal PrP isoform designated PrP^Sc accumulates, and much evidence argues that it is a major and necessary component of the infectious prion. Based on the resistance of native PrP^Sc to proteolysis and to digestion with phosphatidylinositol-specific phospholipase C as well as the enhancement of PrP^Sc immunoreactivity after denaturation, we devised in situ immunoassays for the detection of PrP^Sc in cultured cells. Using these immunoassays, we identified the sites of PrP^Sc accumulation in scrapie-infected cultured cells. We also used these immunoassays to isolate PrP^Sc-producing clones from a new hamster brain cell line (HaB) and found an excellent correlation between their PrP^Sc content and prion infectivity titers. In scrapie-infected HaB cells as well as in scrapie-infected mouse neuroblastoma cells, most PrP^Sc was found to be intracellular and most localized with ligands of the Golgi marker wheat germ agglutinin. In one scrapie-infected HaB clone, PrP^Sc also localized extensively with MG-160, a protein resident of the medial-Golgi stack whereas this colocalization was not observed in another subclone of these cells. Whether the sites of intracellular accumulation of PrP^Sc are limited to a few subcellular organelles or they are highly variable remains to be determined. If the intracellular accumulation of PrP^Sc is found in the cells of the central nervous system, then it might be responsible for the neuronal dysfunction and degeneration which are cardinal features of prion diseases.