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 strains operate as dynamic mixtures of a dominant strain and a minor population of substrains. Direct evidence, however, for the existence of preexisting substrains is lacking. Here we show that, using the well-characterized biologically cloned prion strain, DY TME, replication of the dominant strain is required to suppress replication of a minor, yet highly pathogenic substrain, HY TME. Using two complementary physical methods, we show that selective reduction of the dominant strain, in the absence of prion replication, allowed for the emergence of substrains. The identification of preexisting substrains may contribute to the ability of prions to rapidly adapt to new replication environments such as transmission to a new species or replication in the presence of anti-prion drugs.

Automated summary

Prion diseases are fatal neurodegenerative disorders affecting various mammalian species, including humans. Prions consist of disease-specific conformations of prion proteins and show heritable phenotypes under controlled transmission conditions. This study reveals that replicating the dominant strain is necessary to suppress the replication of minor substrains, and selective reduction of the dominant strain allows for the emergence of substrains. Preexisting substrains may contribute to prion strain evolution, drug resistance, and interspecies transmission.