De novo generation of infectious prions with bacterially expressed recombinant prion protein

Abstract

The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

Prions: Beyond a Single Protein

“…In light of these observations, the authors suggested that the GPI anchor of PrP Sc has little or no role in either the propagation or infectivity of prion agents. The finding that prion infectivity does not require the GPI anchor is consistent with observations by several groups, including Chesebro and others mentioned above (98,184,188,189). Importantly, however, the de novo generation of prion infectivity can be achieved with rPrP alone.…”

“…Several recent studies have, indeed, generated transmissible prion diseases with rPrP in natural hosts (96,97,100,189,(191)(192)(193)(194). These infectious recombinant prions were generated with or without the application of serial PMCA (sPMCA) (96,97,99,100,189,(191)(192)(193)(194)(195)(196)(197)(198).…”

Heparan Sulfate and Heparin Promote Faithful Prion Replication in Vitro by Binding to Normal and Abnormal Prion Proteins in Protein Misfolding Cyclic Amplification

“…However, the PrP Sc amplified through PMCA performed using phosphatidylethanolamine alone did not preserve the prion strain characteristics of the input PrP Sc (53). Synthetic phospholipids and RNA induced the spontaneous generation of two types of prions in Escherichia coli recPrP-based PMCA, and their prion strain characteristics appeared to be unchanged during the serial amplification (8,54,55). Our study suggests that polyanions such as GAGs and nucleic acids were essential for accurate in vitro replication of prions in PMCA performed using Bac-PrP.…”

The structure of the infectious prion protein

“…Another promising approach is based on the development of techniques that allow for the efficient production of PrP Sc from recombinant sources in vitro. [67][68][69] H/D exchange in combination with mass spectrometry 70 or solid-state NMR spectroscopy 71 should be able to provide structural information on a local level complementary to the data available by electron microscopy. Prions were found to escape the selection pressure of antiprion compounds in vivo through the selection of drug-resistant, structural variants.…”