PrPSc formation and clearance as determinants of prion tropism.

Prion strains are characterized by strain-specific differences in neuropathology but can also differ in incubation period, clinical disease, host-range and tissue tropism. The hyper (HY) and drowsy (DY) strains of hamster-adapted transmissible mink encephalopathy (TME) differ in tissue tropism and susceptibility to infection by extraneural routes of infection. Notably, DY TME is not detected in the secondary lymphoreticular system (LRS) tissues of infected hosts regardless of the route of inoculation. We found that similar to the lymphotropic strain HY TME, DY TME crosses mucosal epithelia, enters draining lymphatic vessels in underlying laminae propriae, and is transported to LRS tissues. Since DY TME causes disease once it enters the peripheral nervous system, the restriction in DY TME pathogenesis is due to its inability to establish infection in LRS tissues, not a failure of transport. To determine if LRS tissues can support DY TME formation, we performed protein misfolding cyclic amplification using DY PrPSc as the seed and spleen homogenate as the source of PrPC. We found that the spleen environment can support DY PrPSc formation, although at lower rates compared to lymphotropic strains, suggesting that the failure of DY TME to establish infection in the spleen is not due to the absence of a strain-specific conversion cofactor. Finally, we provide evidence that DY PrPSc is more susceptible to degradation when compared to PrPSc from other lymphotrophic strains. We hypothesize that the relative rates of PrPSc formation and clearance can influence prion tropism.

Incongruity between Prion Conversion and Incubation Period following Coinfection.

UNLABELLED: When multiple prion strains are inoculated into the same host, they can interfere with each other. Strains with long incubation periods can suppress conversion of strains with short incubation periods; however, nothing is known about the conversion of the long-incubation-period strain during strain interference. To investigate this, we inoculated hamsters in the sciatic nerve with long-incubation-period strain 139H prior to superinfection with the short-incubation-period hyper (HY) strain of transmissible mink encephalopathy (TME). First, we found that 139H is transported along the same neuroanatomical tracks as HY TME, adding to the growing body of evidence indicating that PrP(Sc) favors retrograde transneuronal transport. In contrast to a previous report, we found that 139H interferes with HY TME infection, which is likely due to both strains targeting the same population of neurons following sciatic nerve inoculation. Under conditions where 139H blocked HY TME from causing disease, the strain-specific properties of PrP(Sc) corresponded with the strain that caused disease, consistent with our previous findings. In the groups of animals where incubation periods were not altered, we found that the animals contained a mixture of 139H and HY TME PrP(Sc) This finding expands the definition of strain interference to include conditions where PrP(Sc) formation is altered yet disease outcome is unaltered. Overall, these results contradict the premise that prion strains are static entities and instead suggest that strain mixtures are dynamic regardless of incubation period or clinical outcome of disease. IMPORTANCE: Prions can exist as a mixture of strains in naturally infected animals, where they are able to interfere with the conversion of each other and to extend incubation periods. Little is known, however, about the dynamics of strain conversion under conditions where incubation periods are not affected. We found that inoculation of the same animal with two strains can result in the alteration of conversion of both strains under conditions where the resulting disease was consistent with infection with only a single strain. These data challenge the idea that prion strains are static and suggests that strain mixtures are more dynamic than previously appreciated. This observation has significant implications for prion adaptation.

Characterization of conformation-dependent prion protein epitopes.

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

Replication efficiency of soil-bound prions varies with soil type.

Prion sorption to soil is thought to play an important role in the transmission of scrapie and chronic wasting disease (CWD) via the environment. Sorption of PrP to soil and soil minerals is influenced by the strain and species of PrP(Sc) and by soil characteristics. However, the ability of soil-bound prions to convert PrP(c) to PrP(Sc) under these wide-ranging conditions remains poorly understood. We developed a semiquantitative protein misfolding cyclic amplification (PMCA) protocol to evaluate replication efficiency of soil-bound prions. Binding of the hyper (HY) strain of transmissible mink encephalopathy (TME) (hamster) prions to a silty clay loam soil yielded a greater-than-1-log decrease in PMCA replication efficiency with a corresponding 1.3-log reduction in titer. The increased binding of PrP(Sc) to soil over time corresponded with a decrease in PMCA replication efficiency. The PMCA efficiency of bound prions varied with soil type, where prions bound to clay and organic surfaces exhibited significantly lower replication efficiencies while prions bound to sand exhibited no apparent difference in replication efficiency compared to unbound controls. PMCA results from hamster and CWD agent-infected elk prions yielded similar findings. Given that PrP(Sc) adsorption affinity varies with soil type, the overall balance between prion adsorption affinity and replication efficiency for the dominant soil types of an area may be a significant determinant in the environmental transmission of prion diseases.