Human prion protein sequence elements impede cross-species chronic wasting disease transmission

Abstract

Chronic wasting disease (CWD) is a fatal prion disease of North American deer and elk and poses an unclear risk for transmission to humans. Human exposure to CWD occurs through hunting activities and consumption of venison from prion-infected animals. Although the amino acid residues of the prion protein (PrP) that prevent or permit human CWD infection are unknown, NMR-based structural studies suggest that the β2-α2 loop (residues 165-175) may impact species barriers. Here we sought to define PrP sequence determinants that affect CWD transmission to humans. We engineered transgenic mice that express human PrP with four amino acid substitutions that result in expression of PrP with a β2-α2 loop (residues 165-175) that exactly matches that of elk PrP. Compared with transgenic mice expressing unaltered human PrP, mice expressing the human-elk chimeric PrP were highly susceptible to elk and deer CWD prions but were concurrently less susceptible to human Creutzfeldt-Jakob disease prions. A systematic in vitro survey of amino acid differences between humans and cervids identified two additional residues that impacted CWD conversion of human PrP. This work identifies amino acids that constitute a substantial structural barrier for CWD transmission to humans and helps illuminate the molecular requirements for cross-species prion transmission.

Figure 5. Atomic space-filling and surface representations of the PrP loop highlight the side chain interactions at the zipper interface.

(A) Atomic space-filling model illustrates the view down the fibril axis. The amino acid side chains of donor cervid PrP (gray) and recipient cervid PrP (white) interdigitate in a class 3 steric zipper. (B) In contrast, the zipper interactions between the donor cervid PrP (gray) and the recipient human PrP (white), which contain yellow side chains (M166, E168, S170, N174), generate a cavity (arrow) between human S170 and cervid N170, as well as steric clash (blue rectangle) between human residue E168 and cervid residue Q168. (C) A side view of the surface of the cervid PrP loop modeled as a β-sheet (red). The interacting β-sheet has been removed to provide a clear view of the interface. A similar side view of the β-sheet surface of the human PrP loop (yellow) reveals cavities near residue S170 in the core of the zipper interface (black rectangle). (D) A magnified, rotated view of the inset in B shows the clash between human residue E168 (yellow) and cervid residue Q168 (red).

Figure 4. Two elk residue substitutions in the β2-α2 loop of human PrP^C enable 100% conversion by CWD prions in a cell lysate–based PMCA.

(A) CWD-seeded conversion of human PrP^C with a single M166V, E168Q, S170N, or N174T substitution showed conversion of HuPrP-168Q and HuPrP-170N. Human PrP^C was not converted by CWD prions. Samples without PK show that PrP^C levels were equivalent. Cer, cervid. (B) Quantification of CWD-seeded human PrP^C variants relative to cervid PrP^C. (C) CWD-seeded conversion of HuPrP-168Q,170N and cervid PrP^C showed similar levels of conversion. (D) Quantification of CWD-seeded human PrP^C variants relative to cervid PrP^C. The conversion of HuPrP-168Q,170N and HuPrP-166V,168Q,170N was not significantly different than that of cervid PrP^C. (E) CWD-seeded conversion of human PrP^C with a single M138L, S143N, H155Y, I184V, or V203I substitution. (F) Quantification of CWD-seeded human PrP^C variants relative to cervid PrP^C. In A and E, the “No PrP^C” lane shows untransfected RK13 cell lysate that was seeded and subjected to PMCA as a control. There was no detection of the seed as shown here. **P < 0.01, 2-tailed, unpaired Student’s t test, relative to conversion of HuPrP (n = 3–6 experimental replicates each).

Figure 3. Tg(HuPrP^elk166–174)-CWD prions display increased stability in chaotropes and resistance to enzyme degradation as compared with elk CWD.

PrP^Sc stability as assessed by guanidine hydrochloride (GdnHCl) denaturation was significantly greater in Tg(HuPrP^elk166–174) mice than in elk. (A) Western blots and (B) denaturation curves show a representative example from four independent experiments. (C) The bar graph shows the GdnHCl concentration at which half the PrP has been PK-digested ([GdnHCl]_1/2) (n = 4 mice and 4 replicates of one elk); results are from four experiments (mean ± SEM). (D) PrP^Sc was separated from PrP^C by size exclusion chromatography, then samples were split and either treated or not treated with PK and analyzed by Western blot. (E) Quantification of blots shows that significantly more PrP^Sc is PK resistant in Tg(HuPrP^elk166–174) mice than in elk. (F) Brain homogenates from Tg(HuPrP) mice inoculated with sCJD prions show PK-resistant PrP^Sc by Western blot. (G) Brain homogenates from Tg(HuPrP^elk166–174) mice inoculated with sCJD or CWD prions show different PK-resistant PrP^Sc migration patterns. **P < 0.01, 2-tailed, unpaired Student’s t test.

Figure 2. CWD prions differ biochemically in Tg(HuPrP^elk166–174) mice as compared with cervid CWD or human CJD.

(A) PrP^Sc from Tg(HuPrP^elk166–174) brain migrates faster and has a glycoform pattern (ratio of di-, mono-, and un-glycosylated PrP^Sc) different from that of elk CWD by Western blot analysis. Results for six of the seven positive mice are shown here. Red dashes indicate migration of unglycosylated PrP^Sc. (B) In Tg(HuPrP) mice inoculated with elk CWD, the brain shows no detectable PrP^Sc, even after NaPTA precipitation. (C) Tg(HuPrP^elk166–174) mice infected with deer CWD show a PrP^Sc electrophoretic migration and glycoform pattern indistinguishable from that of Tg(HuPrP^elk166–174) mice inoculated with elk CWD. (D) As in B, no Tg(HuPrP) mice inoculated with deer CWD have detectable PrP^Sc in the brain. (E) Sub-passaged CWD-Tg(HuPrP^elk166–174) (P2) in Tg(HuPrP^elk166–174) mice revealed no change in the electrophoretic mobility or glycoform pattern of PrP^Sc as compared with PrP^Sc from P1. (F) Comparison of Tg(HuPrP^elk166–174)-CWD prions with human sporadic and variant CJD prions. Isolates consisted of type 1 or type 2 sporadic CJD prions from individuals homozygous for methionine (MM) or valine (VV) at PRNP codon 129; Tg CWD, CWD-infected Tg(HuPrP^elk166–174). (G) Measurements of di-, mono-, and un-glycosylated PrP^Sc from CWD-inoculated Tg(HuPrP^elk166–174) mice differed significantly from those of CWD-infected elk and deer. **P < 0.01; 2-tailed, unpaired Student’s t test comparing ratio of di- and monoglycosylated PrP (n = 3–6 animals per group).

Figure 1. Mice expressing a human-elk chimeric PrP^C are infected by CWD prions.

(A) Human PrP^C sequence with elk residue differences shown below. The human residue Q223 is also present in mule deer, but is E223 in elk. Amino acid substitutions present in the Tg(HuPrP^elk166–174) mice are in red. (B) Neurologic signs in CWD-inoculated Tg(HuPrP^elk166–174) mice included hind limb clasp (arrow) typical of prion disease, whereas the hind limb splay of Tg(HuPrP) mice was normal. (C) Kaplan-Meier survival curves of CWD-inoculated Tg(HuPrP^elk166–174) mice reveal a significant decrease in the incubation period on second passage. One mouse died with intercurrent disease at 109 dpi. No Tg(HuPrP) mice developed clinical signs of infection after CWD inoculation. Prion infection status was confirmed by biochemical and histologic assays. P1 and P2, passages 1 and 2. (D) Diffuse PrP^Sc deposition, spongiform degeneration (arrowheads) (H&E), and astrogliosis (GFAP) localize to the thalamus of deer or elk CWD–inoculated Tg(HuPrP^elk166–174) mice, but do not occur in elk CWD–inoculated Tg(HuPrP) mice. Scale bar: 50 μm. (E) The CJD-inoculated Tg(HuPrP) mice manifested neurologic signs, including a stiff tail (arrow), by 173 dpi. (F) Tg(HuPrP) mice inoculated with human sCJD prions developed terminal disease by 186 dpi, whereas Tg(HuPrP^elk166–174) animals developed terminal disease between 260 and 290 dpi. **P < 0.01; ***P < 0.001; log-rank (Mantel-Cox) test.