Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

Abstract

During prion infection, the normal, protease-sensitive conformation of prion protein (PrPC) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrPSc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrPSc in prion-infected brain homogenates as an initiating seed to convert PrPC and trigger the self-propagation of PrPSc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrPSc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrPSc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrPSc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.

Conformational Properties of Prion Strains Can Be Transmitted to Recombinant Prion Protein Fibrils in Real-Time Quaking-Induced Conversion

“…More recently, wild-type mice developed clinical disease typical of TSE at about 130 days after injection of proteinase K (PK)-resistant rPrP fibrils generated by unseeded PMCA in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), a synthetic lipid molecule, and total liver RNA (22). Although these results were reproduced by the same group (23), others have reported that rPrP fibrils generated by the same method were unable to induce either neuropathological changes or the accumulation of PrP Sc (24). Thus, the role of POPG and RNA in the de novo generation of infectious rPrP fibrils remains controversial.…”

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

“…In addition, the interaction between recombinant PrP C and nucleic acids (DNAs, tRNA, and PolyA) simultaneously produces a mixture of condensed and functionally active nucleoprotein complex, PrP Sc oligomers and linear and spherical amyloids. [8][9][10][11][12] A specific nucleic acid as a cofactor for the propagation of prion infection has not been identified. 13 PrP C is a cell surface protein and nucleic acids in extracellular circulation can interact with it.…”

“…16 The presence of prion protein in cytoplasm of cells including neurons has been shown and although the exact biological role of prion proteinnucleic acid interaction is not known at present, it is possible that structural conversion of PrP C to PrP Sc can be catalyzed by cytoplasmic nucleic acids that can play a role in the prion diseases. 12,17,18 A separate study indicates the neurotoxic effect found on the cultured rat cortical neurons of the complex of the ovine prion protein (OvPrP(C)) and RNA. 19,20 Besides, unique quadruplex structure and selective interaction of an RNA aptamer against bovine prion protein and may inhibits the prion disease propagation.…”

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms

“…This refolding parallels the relative ease of producing PrP-res forms, but not infectivity, in a test-tube. 4,[10][11][12] Dilution of the chemical denaturants here did not restore infectivity. While restoration of TSE agent infectivity has been tried for many years in various preparations, 39,55 a small amount of infectivity was recently restored by combining phenol purified 263K brain nucleic acids with recombinant PrP.…”

“…Misfolded recombinant PrP is not reproducibly infectious in animal or culture assays, even though massive amounts of PrPres amyloid are readily generated in a test tube. 4,[10][11][12][13] Huge increases in PrP-res can also coincide with a >5 log loss of FU-CJD agent from chronically infected living cell cultures. 12 This PrP-res is indistinguishable from the "infectious" PrP-res form, yet it is unable to produce any detectable agent in sensitive infectivity culture assays.…”