Interspecies prion transmission often leads to stable changes in physical and biological features of prion strains a phenomenon referred to as a strain mutation. sensitivity to proteolytic digestion and a replication rate of 106-fold/PMCAb round which exceeded that of 263K by almost 104-fold. A series of PMCAb experiments revealed that 263KR+ was lacking in brain-derived 263K material but emerged as a result of changes in RNA content. A similar transformation was also observed for strain Hyper suggesting that this phenomenon was not limited to 263K. The current work demonstrates that dramatic PrPSc transformations can be induced by changes in the prion replication environment and without changes in PrP primary structure.-Gonzalez-Montalban N. Jin Lee Y. Makarava N. Savtchenko R. Baskakov I. V. Changes in prion replication environment cause prion strain mutation. brain-derived PrPSc could be observed as a result of PrPSc amplification in serial PMCA (sPCMA; refs. 17 20 These studies suggest that prion strains might indeed exhibit high levels of conformational plasticity and are subject to transformation when exposed to new replication environments. While ARRY-334543 rare spontaneous strain mutations are believed to be one of the major sources of prion conformational diversity the molecular origin and mechanisms underlying mutations are unknown. Can mutations occur in the absence ARRY-334543 of changes in PrP primary structure? What are the roles of cellular cofactors and the prion replication environment in strain mutations? Previous studies demonstrated that prions replicate with the assistance of other molecules (21). RNAs and polyanions were shown to form a favorable biochemical environment that catalyzes prion replication (3 17 21 -23). Although RNA can serve as a catalyst it does not appear to be an essential component of PrPSc particles (24 25 nor is it important for defining prion strain-specific features (23). Recent studies revealed that lipids might serve as possible cellular cofactors that are important for generating prions with high infectivity titers and for defining strain-specific features (3 6 26 The current study asked the question whether changes in prion replication ARRY-334543 environment and specifically RNA content in the absence of alteration in PrP primary structure lead to a stable change in PrPSc properties. We found that while adaptation of 263K or hyper (HY) PrPSc to an RNA-depleted environment in PMCA with beads (PMCAb) did not change their features PrPSc of both strains underwent remarkable transformation on readaptation to an environment containing RNA. The 263K PrPSc readapted to the RNA-containing environment (referred to as 263KR+) displayed dramatically lower conformational IL-1RAcP stability and proteinase K (PK) resistance than that of 263K. Moreover the PMCAb amplification rate for 263KR+ was found to be 3-4 orders of magnitude higher than that of 263K. Furthermore the current study revealed ARRY-334543 that 263KR+ was lacking in the original brain-derived or PMCAb-derived 263K material but emerged as a result of change in RNA content. The present work demonstrated that changes in the prion replication environment not only created conditions for selective amplification of minor PrPSc conformers but could also give rise to a PrPSc transformation. In other words change ARRY-334543 in replication environment plays an active role in generating prion conformational diversity even in the absence of changes in PrP primary structure. MATERIALS AND METHODS Ethics statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health (NIH). The protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland (assurance number A32000-01: permit number: 0312020). PMCAb Normal brain homogenate (NBH; 10%) from healthy hamsters was prepared as described previously (27) and used as a substrate for PMCAb (28). NBH (10%) in conversion buffer was used as the substrate in PMCAb reactions. To produce RNA-depleted NBH 50 μl of 10 mg/ml RNase A (R4875; Sigma-Aldrich St. Louis MO USA) was added to 5 ml of 10% NBH to a final RNase concentration of 100 μg/ml. To prepare mock-digested NBH 50 μl of RNA-free water was added to 5 ml of 10% NBH. Both mixtures were incubated at 37°C for 1 h under gentle rotation and then total RNA was purified and analyzed using gel electrophoresis. The lack of ARRY-334543 RNA in RNase-treated NBH was confirmed by agarose gel. To prepare seeds 10 scrapie brain homogenates in PBS were serially diluted 102- to 104-fold in conversion.