Fig 1.
Comparison of the susceptibility of Mo BH and BV BH to different PrPSc seeds.
Western blots probed with anti-PrP mAb 27/33 (epitope = 136–158 mouse numbering) showing three-round BH sPMCA reactions. Within each blot, reactions using Mo BH substrate are shown in the top row, and BV BH substrate in the bottom row. Reactions were seeded with various (A) Mo or (B) BV seeds, as indicated. The input seed concentration of all recPrPSc seeds was 6 μg/mL for a final reaction concentration of 0.6 μg/mL. Blots are representative of at least three independent experiments. −PK = sample not subjected to proteinase K digestion; all other samples were proteolyzed. Day 0 = seeded reaction not subjected to sonication. Note that input recPrPSc seeds migrate at a lower MW than the converted native PrPSc sPMCA product, as indicated by the boxed labels.
Fig 2.
Protein-only recPrPSc seeds effectively propagate in BV BH, but not Mo BH sPMCA reactions.
Western blots probed with anti-PrP mAb 27/33. (A) Titration of Mo protein-only recPrPSc in vitro. Western blots showing three-round BH sPMCA reactions with either Mo BH (top row) or BV BH (bottom row) substrates. Reactions were seeded with ten-fold serial dilutions of Mo protein-only recPrPSc. The 10−1 reaction was seeded with 6 μg/mL of Mo protein-only recPrPSc for a final reaction concentration of 0.6 μg/mL of seed. (B) Titration of BV recPrPSc seeds in vitro. Western blots showing the third round of three-round BH sPMCA reactions with either BV BH (left column) or Mo BH (right column) substrates. Reactions were seeded with 10-fold serial dilutions of the indicated BV recPrPSc seed. The 10−1 reactions were seeded with 6 μg/mL of recPrPSc for a final reaction concentration of 0.6 μg/mL. NS- no seed. (C) M109 recPrP amyloid seeded three-round BV BH sPMCA reactions. Reactions were seeded with the increasing concentrations of amyloid, as indicated. Note that input recPrP amyloid seeds migrate at a lower MW than the converted native PrPSc sPMCA product, as indicated by the boxed labels.
Fig 3.
Histopathology of inoculated M109 bank voles.
Representative microscopic images of brain sections of M109 bank voles stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry (IHC) with anti-PrP mAb 27/33, as indicated. Rows from top to bottom: asymptomatic control bank vole sacrificed 410 days after inoculation with a 10−1 dilution of the original 6 μg/mL recombinant sPMCA input seed (Mo cofactor recPrPSc) serially diluted 1:10 eighteen times in recombinant sPMCA reaction buffer to demonstrate that there is no remaining infectivity from the input seed; terminally ill bank vole sacrificed 134 days after inoculation with a 10−1 dilution of BV M109 cofactor recPrPSc (final concentration = 0.6 μg/mL); terminally ill bank vole sacrificed 99 days after serial passage of BV M109 cofactor recPrPSc (10−1 dilution of 10% w/v BH); asymptomatic bank vole sacrificed 403 days after inoculation with 10−1 dilution BV M109 protein-only recPrPSc (final concentration = 0.6 μg/mL); and a terminally ill bank vole sacrificed 113 days after inoculation with [protein-only→BH PrPSc] (10−1 dilution of round three of the BH sPMCA reaction). The inoculum volume used was 30 μL. Scale bar = 100 μm.
Fig 4.
Proteinase K digestion of control and experimentally infected bank voles.
(A) Western blots probed with anti-PrP mAb 27/33 showing PrPSc in brain homogenates from M109 bank voles from the indicated control or experimental condition. Top Panel: BH aliquots treated with 64 μg/mL PK (+PK). Bottom panel: BH aliquots that were not subjected to PK digestion (-PK). (B) Western blots comparing the protease-resistance levels of PrPSc in brain homogenates from M109 bank voles from various control or experimental conditions, as indicated. Samples were digested with various concentrations of PK for 1 hr at 37 oC, as indicated. 2° = passage of the experimental sample.
Table 1.
sPMCA using recombinant PrP substrate inoculations in M109 genotype bank voles.
Table 2.
Inoculations in Mice.
Table 3.
[protein-only→BH PrPSc] inoculations into M109 bank voles.
Fig 5.
Regional neuropathology of M109 cofactor recPrPSc and [protein-only→BH PrPSc] infected bank voles.
Profiles of vacuolation scores of animals inoculated with either M109 cofactor recPrPSc (orange squares) or [protein-only→BH PrPSc] (blue circles). Mean values ± SEM are shown. N = 6 for all measurements except for [protein-only→BH PrPSc] cerebellum and pons, where N = 3.
Fig 6.
Protein-only recPrPSc seeds require cofactor molecules to convert immunopurified BV PrPC to PrPSc.
Western blots probed with anti-PrP mAb 27/33. Immunopurified M109 BV PrPC substrate was supplemented with, from left to right, PrP0/0 BH, RNA, purified lipid cofactor, or PBS and 1% Triton X-100 buffer (-cofactor), as indicated. All of the reconstituted reactions were then seeded with BV M109 protein-only recPrPSc (top), or Mo protein-only recPrPSc (bottom), and subjected to three-round sPMCA.
Table 4.
Inoculations of M109 protein-only rec.
PrPSc-seeded sPMCA reactions using recPrP substrate and cofactor molecules into M109 bank voles.
Fig 7.
Unified model of mammalian prion infectivity.
Proposed model of prion infectivity, in which the global structure of protein-only PrPSc (formed in reaction I lacking cofactor molecules) can store latent information, but local conformational changes caused by the absence of cofactor abrogates infectivity. The local changes can be repaired by sPMCA in substrate containing BV PrPC and cofactors (reaction II), immediately restoring full specific infectivity. Despite the temporary loss of infectivity, the two-step process (reactions I + II) recovers a prion strain that possesses full specific infectivity and is clinically, biochemically, and pathologically indistinguishable from BV cofactor recPrPSc, in which the specific infectivity of the parental seed was continuously maintained by propagation in the presence of cofactor molecules (reaction III). Non-infectious, protein-only samples are shown in blue, and infectious samples produced with cofactor are shown in red.