Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification

Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.     

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP^Sc. Studies showed that recombinant cellular PrP, PrP^C, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V₁₃₆L₁₄₁R₁₅₄Q₁₇₁ and A₁₃₆F₁₄₁R₁₅₄Q₁₇₁, and one genotype of white-tailed deer (Q₉₅G₉₆, X₁₃₂,Y₂₁₆) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP^C shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP^C and supports amplification of PrP^Sc and PrP^CWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP^Sc or PrP^CWD production. Addition of 2 to 20 μM of manganese chloride (MnCl₂) to unseeded PMCA resulted in conversion of recombinant PrP^C to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.     

In vitro self-propagation of recombinant PrPSc-like conformation generated in the yeast cytoplasm

Self-propagation is characteristic property for a prion conformation. Previous studies revealed that prion protein expressed in the cytoplasm gained a PrP(Sc)-like conformation. However, it remains unclear whether the PrP(Sc)-like conformation has the self-propagating property. We found that PrP partially purified from yeast cytoplasm formed amyloid fiber like structures, and we found that the PrP(Sc)-like conformation is able to convert normal PrP(C) in the brain homogenate to a proteinase K-resistant conformation. These results suggest that yeast cytoplasm expressed recombinant PrP(Sc)-like conformation has the characteristic self-propagating property of a prion, which may have implications in the pathogenesis of sporadic and inherited prion diseases.