Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

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Abstract
Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrPC molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrPC is pre-incubated with poly(A) RNA and PrPSc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrPSc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrPSc molecules, most likely through an epitope containing residue 172. Over the past two decades, various investigators have observed that heterozygous animals possessing two different forms of the gene encoding the prion protein (PrP) are more difficult to infect with some strains of infectious prions than homozygous animals possessing only the most commonly occurring form of the gene encoding PrP for that species. In 1995, it was hypothesized that the inhibition of prion infection in heterozygous animals might be caused by competition between the two different types of PrP molecules for binding to a common cofactor required for prion propagation, provisionally named “protein X,” through a specific portion of the PrP molecule. Here, we report that mixing different purified PrP molecules together in test tube reactions lacking accessory proteins can also interfere with prion propagation. We also found that some mutations of the putative protein X binding site do not inhibit the formation of hamster prions in chemical reactions. Our work suggests that different PrP molecules most likely compete for binding to newly formed prions rather than an accessory protein cofactor, and argues against the existence of protein X.