Spontaneous generation of distinct prion variants with recombinant prion protein from a baculovirus-insect cell expression system

Prion diseases are transmissible and progressive neurodegenerative disorders characterized by abnormal prion protein (PrPSc) accumulation in the central nervous system. Generation of synthetic PrPSc in a cell-free conversion system and examination of its transmissibility to animals would facilitate testing of the protein-only hypothesis and the understanding of the molecular basis of sporadic prion diseases. In this study, we used recombinant prion protein from a baculovirus-insect cell expression system (Bac-rPrP) and insect cell-derived cofactors to determine whether Bac-rPrP(Sc) is spontaneously produced in intermittent ultrasonic reactions. No spontaneous generation of Bac-rPrP(Sc) was observed at 37 degrees C, but when the reaction temperature was increased to 45 degrees C, Bac-rPrP(Sc) was generated in all trials. Some BacrPrP(Sc) variants were transmissible to mice, but when the reaction was repeated for 40 rounds, the transmissibility was lost. Notably, a variety of Bac-rPrP(Sc) variants, including non-transmissible ones, differing in resistance to proteinase K and cofactor dependence during amplification, was generated under the same experimental conditions, including the same sonication settings and cofactors. However, their characteristics also disappeared after 40 reaction rounds and the variety converged onto a single variant. These results indicate that various Bac-rPrP(Sc) variants with different transmissibility to mice and structural properties are generated, which compete with each other and gradually converge onto a variant with a slightly faster amplification rate. (C) 2022 Elsevier Inc. All rights reserved.