Insights into the Bidirectional Properties of the Sheep-Deer Prion Transmission Barrier

Abstract

The large chronic wasting disease (CWD)-affected cervid population in the USA and Canada, and the risk of the disease being transmitted to humans through intermediate species, is a highly worrying issue that is still poorly understood. In this case, recombinant protein misfolding cyclic amplification was used to determine, in vitro, the relevance of each individual amino acid on cross-species prion transmission. Others and we have found that the β2-α2 loop is a key modulator of transmission barriers between species and markedly influences infection by sheep scrapie, bovine spongiform encephalopathy (BSE), or elk CWD. Amino acids that differentiate ovine and deer normal host prion protein (PrP^C) and associated with structural rigidity of the loop β2-α2 (S173N, N177T) appear to confer resistance to some prion diseases. However, addition of methionine at codon 208 together with the previously described rigid loop substitutions seems to hide a key in this species barrier, as it makes sheep recombinant prion protein highly susceptible to CWD-induced misfolding. These studies indicate that interspecies prion transmission is not only governed just by the β2-α2 loop amino acid sequence but also by its interactions with the α3-helix as shown by substitution I208M. Transmissible spongiform encephalopathies, characterized by long incubation periods and spongiform changes associated with neuronal loss in the brain, have been described in several mammalian species appearing either naturally (scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids, Creutzfeldt-Jakob disease in humans) or by experimental transmission studies (scrapie in mice and hamsters). Much of the pathogenesis of the prion diseases has been determined in the last 40 years, such as the etiological agent or the fact that prions occur as different strains that show distinct biological and physicochemical properties. However, there are many unanswered questions regarding the strain phenomenon and interspecies transmissibility. To assess the risk of interspecies transmission between scrapie and chronic wasting disease, an in vitro prion propagation method has been used. This technique allows to predict the amino acids preventing the transmission between sheep and deer prion diseases.

Keywords: Chronic wasting disease (CWD); In vitro propagation; PMCA; Prion diseases; Scrapie; Transmissible spongiform encephalopathy (TSE); Transmission barrier.

Fig. 1

a Graphic representation of PrP^res showing tubes in rounds R05 to R20 of serial rec-PMCA using sheep and mule deer recombinant proteins as substrate. A substrate mixture consisting of ovine or cervine rec-PrP, complemented with PRNP^−/− brain homogenate and seeded with a panel of TSE agents, including cattle BSE, CWD, and different sheep scrapie prions (Dawson, Langlade, UKA2, Sc21) or unseeded were subjected to 20 rounds of rec-PMCA. The percentage of duplicate tubes showing PK-resistant misfolded rec-PrP seen in each round is indicated in a greyscale. b Western blot of different ovine rec-PrP^res (OvDawson rec-PrP^res, OvLanglade rec-PrP^res, OvUKA2 rec-PrP^res, OvSc21 rec-PrP^res, OvBSE rec-PrP^res, OvBSE-H rec-PrP^res, and OvBSE-L rec-PrP^res) and cervine rec-PrP^res (MuDawson rec-PrP^res, MuBSE rec-PrP^res, MuBSE-H rec-PrP^res, MuBSE-L rec-PrP^res, and MuCWD rec-PrP^res) generated in vitro after the 20th serial round of rec-PMCA using ovine and cervine recombinant proteins as substrates. The presence of rec-PrP^res was determined by subjecting the rec-PMCA product to 25 μg/ml PK digestion for 1 h at 42 °C, followed by Western blot analysis using 9A2 antibody (diluted 1:4000). Recombinant ovine or cervine PrPs (ovine rec-PrP and Mu-deer rec-PrP) and tg338 brain homogenate (Tg338 ctrl) were used as controls. PK, Proteinase K; MW, molecular weight

Fig. 2

In vitro propagation of misfolded prion proteins. a Ovine (OvDawson rec-PrP^res, OvLanglade rec-PrP^res, OvUKA2 rec-PrP^res, OvSc21 rec-PrP^res, OvBSE rec-PrP^res, OvBSE-H rec-PrP^res, and OvBSE-L rec-PrP^res) and b cervine (MuDawson rec-PrP^res, MuBSE rec-PrP^res, MuBSE-H rec-PrP^res, MuBSE-L rec-PrP^res, and MuCWD rec-PrP^res) misfolded PK-resistant proteins were diluted from 10⁻² to 10⁻⁸ and amplified by rec-PMCA using ovine ARQ rec-PrP and mule deer rec-PrP as substrates. The negative control samples (0) were either unseeded ovine ARQ rec-PrP or cervine rec-PrP substrates. NA indicates the no-amplification sample which was diluted 10⁻¹ but not subjected to PMCA. The products of a single PMCA round (48 h) were digested with PK (25 μg/ml) for 1 h at 42 °C, analyzed by Western blot and developed with 9A2 antibody (diluted 1:4000). Recombinant ovine or cervine PrPs (rec-PrP) and tg338 brain homogenate (Tg338) were used as controls. PK, Proteinase K; MW, molecular weight. Below the panel of Western blots is a representative experiment from three replicates, maximum dilutions for each strain are plotted as an average of three independent experiments, including standard deviations

Fig. 3

a Sequence alignment of the C-terminal domain (residues 134–234) of ovine PrP compared with mule deer PrP. Note the four amino acid differences that where substituted giving rise to four mutants: ovine ARQ PrP (S98T), ovine ARQ PrP (S173N), ovine ARQ PrP (N177T), and ovine ARQ PrP (I208M). b Diagram of ovine PrP, and the locations of the native secondary structures in sheep ARQ PrP (134–234) are indicated: the α-helical regions are represented in blue and the β-sheet region in red. The diagram was generated using the program Swiss-PdbViewer (4.1.0)

Fig. 4

Evaluation of the effects of substitutions based on cervine PrP on the in vitro propagation of ovine (OvDawson rec-PrP^res and OvBSE rec-PrP^res) and cervine (MuCWD rec-PrP^res) misfolded proteins. Western blots showing PK-resistant PrP from 10⁻² to 10⁻⁸ serial dilutions of OvDawson, OvBSE, and MuCWD rec-PrP^res-seeded rec-PMCA propagation reactions containing wild-type (ovine ARQ), ARQ (S98T), ARQ (S173N), ARQ (N177T), and ARQ (I208M) rec-PrP substrates. For each seed, substrates containing ovine PrP substitutions were complemented with chicken brain homogenate and subjected to one round of rec-PMCA. The negative control samples (0) were either unseeded ovine ARQ rec-PrP or cervine rec-PrP substrate. NA indicates the no-amplification sample which was dilutes 10⁻¹ but not subjected to PMCA. The products of a single PMCA round (48 h) were digested with PK (25 μg/ml) for 1 h at 42 °C, analyzed by Western blot, and developed with 9A2 antibody (diluted 1:4000). Recombinant ovine or cervine PrPs (rec-PrP) and tg338 brain homogenate (Tg338) were used as controls. In all blots, a sample containing wild-type ovine (ARQ rec-PrP) and Tg338 substrate not subjected to PK digestion is shown in the last lane. PK, Proteinase K; MW, molecular weight. Below the panel of Western blots is a representative experiment from three replicates, maximum dilutions for each strain are plotted as an average of three independent experiments, including standard deviations. The Western blots and results corresponding to N177T substitution are also presented in the next figure due to its relevant blocking effect in order to facilitate the interpretation of results in both figures

Fig. 5

In vitro propagation ability of ovine and cervine recombinant misfolded seeds on substrates containing ovine rec-PrP with substitutions that define the cervine rigid loop (S173N T177N) and with other mule deer substitutions in combination with the ones defining the rigid loop. Western blots showing PK-resistant misfolded rec-PrP after serial dilutions from 10⁻² to 10⁻⁸ of OvDawson, OvBSE, and MuCWD rec-PrP^res and a single 48 h rec-PMCA round on substrates containing ovine rec-PrP with substitutions (N177T), (S173N N177T), (S98T N177T), (I208M N177T), (S98T S173N N177T), and (I208M S173N N177T) complemented with chicken brain homogenate. The negative control samples (0) were unseeded ovine ARQ rec-PrP substrate. NA indicates the no-amplification sample with the inocula diluted 10⁻¹ and not subjected to sonication. The rec-PMCA products were digested with PK (25 μg/ml) for 1 h at 42 °C, analyzed by Western blot, and probed with 9A2 antibody (diluted 1:4000). Recombinant ovine or cervine PrPs (rec-PrP) and tg338 brain homogenate (Tg338) were used as controls. PK, Proteinase K; MW, molecular weight. Below the panel of Western blots is a representative experiment of three replicates, and maximum dilutions for each strain are plotted as an average of three independent experiments, including standard deviations. The Western blots and results corresponding to N177T substitution are also presented in the previous figure due to its relevant blocking effect in order to facilitate the interpretation of results in both figures

Fig. 6

a Ribbon diagram of the C-terminal domain of ovine PrP-ARQ (residues 114–234, pdb 1TPX). α-helices in blue and β-sheets in red. Amino acid residues involved in the structural arrangement of β2–α2 loop and α-helix 3 (I208M) are indicated. Hydrogen bonds are shown as dashed black lines. The disulfide bridge is represented by the yellow connection between α-helix 2 and 3. The diagram was generated using the software Swiss-PdbViewer v4.1.0. b The flexible ovine β2–α2 loop (gray) versus the cervine rigid loop β2–α2 (red) (adapted from [47]). c Prion structural dynamics and mechanism of separation of β1–α1–β2 from α2-α3 in relation to the polymerization process (adapted from [91])