Nucleic acids in prion preparations: unspecific background or essential component?

Abstract

As recently published (Kellings et al. J. gen Vir. 73, 1025-1029 (1992)), the analysis of purified scrapie prions by return refocusing gel electrophoresis revealed remaining nucleic acids in the size range up to 1100 nucleotides. The results defined the possible characteristics of a hypothetical scrapie-specific nucleic acid. If homogeneous in size, such a molecule would be less than 80 nucleotides in length at a particle-to-infectivity ratio (P:I) near unity; if heterogeneous, scrapie-specific nucleic acids would have to include molecules smaller than 240 nucleotides. To decrease the amount of nucleic acids, several modifications of the PrPSc purification scheme were introduced. Instead of sucrose gradient, ultrafiltration was applied as a purification step and nucleic acids were degraded by Benzonase after ultrafiltration, but significant reduction of the P:I ratio could not be achieved. To prevent trapping of nucleic acids in prion rods, nuclease (Benzonase) was added into the tissue homogenate and incubated at 37 degrees C, overnight. The Benzonase treatment revealed no loss of infectivity, but the whole procedure of nucleic acid analysis did not lead to a reduction of the P:I ratio. In another approach the number of nucleic acid degradations steps was reduced to essentially two steps: Zn2+ hydrolysis and Benzonase digestion. Higher Zn2+ concentrations and prolonged incubation times resulted in a more efficient nucleic acid degradation. The bioassays yielded complete recovery of infectivity. Large-scale preparations for determining the P:I ratio are still underway.

Protease-Sensitive Scrapie Prion Protein in Aggregates of Heterogeneous Sizes

“…The transmissible spongiform encephalopathies (TSE) such as Creutzfeldt−Jakob disease of humans and scrapie of sheep are caused by prions () (for reviews, see 2 − 4 ). TSE prions seem to propagate by refolding the membrane glycoprotein PrP C into a pathogenic ‘prion' conformation termed PrP Sc , which in turn is their only known component ( , ). These two PrP isoforms thus possess the same covalent structure (), but differ in their conformation as PrP Sc is enriched in β-sheets ( 8, 9 ) while PrP C is predominantly α-helical.…”

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2

“…But both UV and ionizing radiation inactivation studies as well as physical studies have eliminated the possibility of a large nucleic acid hiding within purified preparations of prions (110)(111)(112). Only oligonucleotides of fewer than 50 bases were found at a concentration of one molecule per ID 50 unit in prion preparations highly enriched for scrapie infectivity (113,114). These small nucleic acids were of variable length and are thought to be degradation byproducts generated during purification of prions.…”

Copper(II)-induced Conformational Changes and Protease Resistance in Recombinant and Cellular PrP

“…They are inferred to exist from analyses of the variable neuropathology of sCJD (9,10) and, more compellingly, from the passage properties of prions isolated from sheep with natural scrapie (11). The existence of strains of scrapie prions was widely interpreted to indicate the presence of a nucleic acid genome (12); however, biochemical analyses have failed to provide strong evidence for such an entity (13). More recent studies indicate that strains are associated with PrP Sc variants that can be distinguished by protease cleavage sites in the vicinity of codon 90 (14,15) or the accessibility of residues 104 -113 to the 3F4 antibody (16,17).…”