Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID 50 ) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP Sc molecules and ID 50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID 50 unit is Ϸ25 nucleotides in length. In the same fractions, there were Ϸ3,000 protease-resistant PrP Sc molecules per ID 50 unit after accounting for clearance of PrP Sc following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of Ϸ1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.

Although increasingly unlikely, the possibility of a scrapie-specific nucleic acid carried by infectious prion particles is still unresolved. Return refocusing gel electrophoresis was developed to detect homogeneous and heterogeneous nucleic acids extracted from highly purified scrapie prion preparations. This method was improved with respect to the size range from 13 to 1100 nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small DNA oligonucleotides and polyadenylated RNA, was determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some small polynucleotides remained. Although a scrapiespecific nucleic acid cannot be excluded, the results further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a molecule would be < 80 nt in length at a particle-toinfectivity ratio near unity, if heterogeneous, scrapiespecific nucleic acids would have to include molecules smaller than 240 nt.

An abnormal isoform of the prion protein (PrP) designated PrP Sc is the major, or possibly the only, component of infectious prions. Structural studies of PrP Sc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrP Sc. After ultracentrifugation at 100,000 ؋ g for 1 h, ϳ30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of ϳ6S, ϳ10 nm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% ␣-helical content; addition of 25% acetonitrile induced aggregates high in ␤ sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.

No 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.