Inhibition of Protease-Resistant Prion Protein Formation in a Transformed Deer Cell Line Infected with Chronic Wasting Disease

摘要

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP^CWD) was used as an indicator of CWD infection. Although no PrP^CWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP^CWD-positive clone out of 51. This clone, designated MDB^CWD, has maintained stable PrP^CWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP^CWD-positive subclones out of 30, one of which was designated MDB^CWD2. The MDB^CWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP^CWD accumulation in MDB^CWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP^CWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.