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Prion replication results from the autocatalytic templated assisted conversion of the host-encoded prion protein PrP C into misfolded, polydisperse PrP Sc conformers. Structurally distinct PrP Sc conformers can give rise to multiple prion strains. Within and between prion strains, the biological activity (replicative efficacy and specific infectivity) of PrP Sc assemblies is size dependent and thus reflects an intrinsic structural heterogeneity. The contribution of such PrP Sc heterogeneity across species prion adaptation, which is believed to be based on fit adjustment between PrP Sc template(s) and host PrP C , has not been explored. To define the structural-to-fitness PrP Sc landscape, we measured the relative capacity of size-fractionated PrP Sc assemblies from different prion strains to cross mounting species barriers in transgenic mice expressing foreign PrP C . In the absence of a transmission barrier, the relative efficacy of the isolated PrP Sc assemblies to induce the disease is like the efficacy observed in the homotypic context. However, in the presence of a transmission barrier, size fractionation overtly delays and even abrogates prion pathogenesis in both the brain and spleen tissues, independently of the infectivity load of the isolated assemblies. Altering by serial dilution PrP Sc assembly content of non-fractionated inocula aberrantly reduces their specific infectivity, solely in the presence of a transmission barrier. This suggests that synergy between structurally distinct PrP Sc assemblies in the inoculum is requested for crossing the species barrier. Our data support a mechanism whereby overcoming prion species barrier requires complementation between structurally distinct PrP Sc assemblies. This work provides key insight into the "quasispecies" concept applied to prions, which would not necessarily rely on prion substrains as constituent but on structural PrP Sc heterogeneity within prion population.

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJD V180I ) have been found to share a unique pathological prion protein (PrP Sc ) that lacks the protease-resistant PrP Sc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrP Sc from their cellular PrP (PrP C ). To investigate the seeding activity of these unique PrP Sc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrP C substrates. We observed that the seeding of PrP Sc from VPSPr or fCJD V180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrP Sc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrP Sc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrP C from the mixture of normal TgMM and Tg mouse brain expressing PrP V180I mutation (Tg180) but not TgV180I alone was converted into PrP Sc by seeding with the VPSPr or fCJD V180I . The RT-QuIC seeding activity of PrP Sc from VPSPr and fCJD V180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrP Sc does not affect the glycoforms of in vitro newly amplified PrP Sc .