Evidence for preexisting prion substrain diversity in a biologically cloned prion strain.

Prion diseases are a group of inevitably fatal neurodegenerative disorders affecting numerous mammalian species, including Sapiens. Prions are composed of PrPSc, the disease specific conformation of the host encoded prion protein. Prion strains are operationally defined as a heritable phenotype of disease under controlled transmission conditions. Treatment of rodents with anti-prion drugs results in the emergence of drug-resistant prion strains and suggest that prion strains are comprised of a dominant strain and substrains. While much experimental evidence is consistent with this hypothesis, direct observation of substrains has not been observed. Here we show that replication of the dominant strain is required for suppression of a substrain. Based on this observation we reasoned that selective reduction of the dominant strain may allow for emergence of substrains. Using a combination of biochemical methods to selectively reduce drowsy (DY) PrPSc from biologically-cloned DY transmissible mink encephalopathy (TME)-infected brain resulted in the emergence of strains with different properties than DY TME. The selection methods did not occur during prion formation, suggesting the substrains identified preexisted in the DY TME-infected brain. We show that DY TME is biologically stable, even under conditions of serial passage at high titer that can lead to strain breakdown. Substrains therefore can exist under conditions where the dominant strain does not allow for substrain emergence suggesting that substrains are a common feature of prions. This observation has mechanistic implications for prion strain evolution, drug resistance and interspecies transmission.

Prion protein amino acid sequence influences formation of authentic synthetic PrPSc.

Synthetic prions, generated de novo from minimal, non-infectious components, cause bona fide prion disease in animals. Transmission of synthetic prions to hosts expressing syngeneic PrPC results in extended, variable incubation periods and incomplete attack rates. In contrast, murine synthetic prions (MSP) generated via PMCA with minimal cofactors readily infected mice and hamsters and rapidly adapted to both species. To investigate if hamster synthetic prions (HSP) generated under the same conditions as the MSP are also highly infectious, we inoculated hamsters with HSP generated with either hamster wild type or mutant (ΔG54, ΔG54/M139I, M139I/I205M) recombinant PrP. None of the inoculated hamsters developed clinical signs of prion disease, however, brain homogenate from HSPWT- and HSPΔG54-infected hamsters contained PrPSc, indicating subclinical infection. Serial passage in hamsters resulted in clinical disease at second passage accompanied by changes in incubation period and PrPSc conformational stability between second and third passage. These data suggest the HSP, in contrast to the MSP, are not comprised of PrPSc, and instead generate authentic PrPSc via deformed templating. Differences in infectivity between the MSP and HSP suggest that, under similar generation conditions, the amino acid sequence of PrP influences generation of authentic PrPSc.