Nucleic acid-free mutation of prion strains

Abstract

While prions share the ability to propagate strain information with nucleic acid-based pathogens, it is unclear how they mutate and acquire fitness in the absence of this informational component. Because prion diseases occur as epidemics, understanding this mechanism is of paramount importance for implementing control strategies to limit their spread and for evaluating their zoonotic potential. Here we review emerging evidence indicating how prion protein primary structures, in concert with PrP^Sc conformational compatibility, determine prion strain mutation.

Prion strain mutation has been reported following interspecies transmission, for example transmission of sheep scrapie to rodents.¹ Such circumstances may result in novel strains with an expanded host range.² Chronic wasting disease (CWD), a burgeoning epidemic of cervids, is of particular concern. The only recognized prion disease of wild animals, its unparalleled transmission efficiency exemplifies prion contagion and complicates strategies for its control. Originally described in captive mule deer (Odocoileus hemionus hemionus) in northern Colorado in 1978³ and subsequently in free-ranging mule deer and Rocky Mountain elk (Cervus elaphus nelsoni) in southeastern Wyoming and northeastern Colorado, CWD is emergent in wild and/or farm-raised cervids from 15 states and two provinces in North America. A retrospective assessment also revealed CWD infection of mule deer and black-tailed deer (Odocoileus hemonius columbianus) resident at the Toronto Zoo between 1973 and 2003.⁴ Outbreaks have also occurred in South Korea as a result of importation of sub-clinically infected animals.^5,6 In addition to its increased geographic spread, the known host-range of CWD is also expanding. Since 2002, CWD has emerged in free-ranging populations of white-tailed deer (Odocoileus virginianus) east of the Mississippi.⁷ Most recently CWD has occurred in wild⁸ and captive moose (Alces alces shirasi)⁹ and has been transmitted to European red deer (Cervus elaphus elaphus).¹⁰ Whether the host range of CWD extends beyond the family Cervidae is currently unclear.

While it is now clear to most investigators that prions encipher strain information in the conformation of PrP^Sc,^11–16 the existence and mutability of strains persuaded early researchers that prion diseases were caused by viruses, albeit ones with unconventional properties. The initial framework for understanding prion strains derived from studies of rodent-adapted sheep scrapie isolates, which led to standard criteria for characterizing and differentiating strains, including differences in the distribution and severity of PrP-associated pathology, often revealed by staining brain sections with anti-PrP antibodies¹⁷ and the time to onset of disease after inoculation, referred to as the incubation time.^18,19 Using consistent doses and routes of inoculation, these studies demonstrated the remarkably consistent replication of strains, reflected in incubation times with standard errors generally <2% of the mean when serially passaged in mice of defined genotype.²⁰ Subsequent studies revealed Prnp as the most important host factor controlling incubation time.²¹

By varying transmission conditions, these early studies also identified three classes of strains with varying stabilities.¹⁹ While the properties of class I strains are stable across genotypes, class II strains are stable when passaged in the Prnp genotype in which they were isolated, but gradually change during serial passage in another genotype until achieving a new set of stable properties. Class III strains are stable when passaged at high dilutions, otherwise strain properties break down, giving rise to new properties. While mutational events in agent-associated nucleic acid were originally cited as the cause of strain instability,¹ more recently, changes in the conformation of PrP^Sc were shown to be associated with the acquisition of new strain properties.¹⁵ To account for the phenomena of prion transmission barriers, strain instability, heterogeneity and adaptation in the context of PrP^Sc conformation, the conformational selection model postulates that only a subset of PrP^Sc conformations is compatible with each individual PrP primary structure.^22,23

While strain diversity is well documented for sheep scrapie, BSE, transmissible mink encephalopathy (TME) and human prions, until recently the prevalence of cervid prion strains had not been assessed. Prototype transgenic (Tg) mice expressing deer PrP, designated Tg(CerPrP)1536^+/-24, recapitulated the cardinal neuropathological, clinical and biochemical features of CWD, an observation subsequently confirmed in comparable Tg mouse models.^25–30 Although our original studies²⁴ and subsequent work^26,31 raised the possibility of CWD strain variation, the limited number of isolates and the lack of detailed strain analyses in those studies meant that this hypothesis remained speculative. Subsequent studies supported the feasibility of using Tg(CerPrP)1536^+/- mice for characterizing naturally occurring CWD strains, CWD prions generated by protein misfolding cyclic amplification (PMCA) and novel cervid prions.³² Our comparative studies of CWD in Tg mice expressing deer and elk PrP³⁰ also identified residue 226, the sole primary structural difference between deer and elk PrP, as a major determinant of CWD pathogenesis.

In a recent study, we used Tg(CerPrP) mice to determine the prevalence of CWD prion strains in a greatly expanded collection of captive and wild cervids from different species and geographic locations and to further assess the role of residue 226 on CWD pathogeneis.³³ Our findings represent the first substantial evidence for two prevalent CWD prion strains. When ∼30 CWD isolates were transmitted to Tg(CerPrP) mice, two distinct neuro-pathological patterns were identified. When grouped together, mice with these distinct neuropathologies were associated with different mean incubation times, allowing us to classify mice as having been affected by one of two strains, referred to as CWD1 and CWD2. Patterns of neuropathological lesions produced in prion diseases are generally symmetrical in coronal sections of affected animals and humans, and this was a feature of the CWD1 strain. The unusual asymmetrical distribution of CerPrP^Sc and pathology in the inoculated hemisphere of mice infected by the CWD2 strain was therefore unexpected, but not unprecedented, since it is also a characteristic of certain scrapie strains such as 87A.³⁴

Remarkably, primary transmissions of CWD prions from elk produced either CWD1 or CWD2 profiles, while transmission of deer inocula favored the production of mixed intra-study incubation times and CWD1 and CWD2 neuropathologies. From this we concluded that elk were infected with either CWD1 or CWD2, while deer brains harbored CWD1/CWD2 strain mixtures. In accordance with these findings, limited transmissions of PMCA-generated elk and deer prions produced only the CWD2 strain and mixed CWD1 and CWD2 responses respectively.

The different primary structures of deer and elk provides a framework for understanding these strain profiles and the hypothesis that CWD strain mutation is governed by the relative stabilities of prion propagation by elk and deer PrP primary structures. Thus, propagation of either strain by CerPrP^C-Q226 in deer brain is unstable, and both strains are manifest as mixtures; the almost exclusive manifestation of CWD1 or CWD2 strains following primary transmissions of elk CWD prions reflects relatively stable strain propagation by CerPrP^C-E226 in elk. Supporting this interpretation, serial passage of prions in the brains of diseased Tg(CerPrP)1536^+/- mice produced strain mixtures, regardless of whether those prions originated in diseased elk or deer. Mutation of CWD1 and CWD2 propagated by CerPrP^C-Q226 is reminiscent of unstable (Class III) scrapie strains such as 87A, and its more stable counterpart, ME7,¹ but with CWD1 and CWD2 representing a novel strain class, with neither being a more stable derivative of the other. Because of the role played by residue 226, the description of a lysine polymorphism at this position in deer³⁵ and its possible role on strain stability may be significant.

These structural considerations are of additional importance since they contribute to emerging evidence that the C-terminal domain, in concert with the loop region between beta2 and alpha helix 2, plays an important role in prion replication. Polymorphic variation resulting in replacement of phenylalanine for serine in mule deer PrP at residue 225, adjacent to the PrP variation in elk and deer, appears to be protective for the development of CWD.³⁶ Residue 226 (222 in mice) was previously implicated in the binding of the hypothetical protein X to PrP.^37,38 Recent structural studies indicating that alpha helix 3 in the C-terminal region affects structural stability of the loop region,³⁹ support previous findings in Tg mice the suggesting the existence of a discontinuous epitope controlling prion propagation.⁴⁰ This long-range interaction is of further interest in light of evidence that rigid or flexible loop structures dictated by amino acid substitutions in loop at MoPrP residues 170 and 174, control interspecies prion transmission.⁴¹

Given the distinct biological properties of CWD1 and CWD2, and the aforementioned proposal that strain characteristics are enciphered in the conformation of PrP^Sc,^11,13–16 assessing the properties of CerPrP^Sc associated with each strain was of considerable interest. The electrophoretic migration patterns of CerPrP^Sc from the brains of mice infected by either strain were indiscernible. CerPrP^Sc associated with CWD1 and CWD2 was composed of equivalent proportions of di-, mono- and a-glycosyl forms and CerPrP^Sc from CWD1- and CWD2-infected mice had similar unfolding characteristics following treatment with guanidinium hydrochloride. The indistinguishable CerPrP^Sc conformations and unstable strain transitions of CWD1 and CWD2 in deer and Tg(CerPrP)1536^+/- mice are consistent with their separation by relatively low energy barriers. Our inability to resolve subtle biochemical properties of CerPrP^Sc is reminiscent of the properties of postulated mutant substrains.⁴² While certain strains are associated with distinct conformers of PrP^Sc,^{11,13,32,43,44} not all strains that can be differentiated by biological means have readily recognizable differences in PrP^Sc.^42,44,45 However, assuming the general protein-only hypothesis is correct, biochemical differences in PrP^Sc must exist. A precondition to resolving their physico-chemical differences of CWD1 and CWD2 is to establish conditions for their stable propagation. We are currently testing the hypothesis that Tg mice expressing elk PrP stably propagate either strain. While strain cloning could be accomplished by re-isolation of CWD1 or CWD2 at the end point of bioassays, biological cloning of cervid prion strains might also be facilitated by our recently described cell culture approaches for studying cervid prions.⁴⁶ CWD1 and CWD2 strains purified by these means may differ with respect to protease-sensitive disease associated PrP (sPrP^Sc) or may be distinguished using more sensitive approaches, including Conformational Dependent Immunoassay,⁴⁷ Fourier Transform Infrared spectroscopy (FT-IR)^48–50 or luminescent conjugated polymers.⁵¹

When two prion strains infect a single host, one strain can interfere with the ability of the other to cause disease. It is unknown whether CWD1 and CWD2 interfere or act synergistically, or whether their co-existence contributes to the unparalleled efficiency of CWD transmission. Interestingly, transmission results reported in previous studies suggested that cervid brain inocula might be composed of strain mixtures.^27,31 Additional previous studies also support our characterization of CWD1 and CWD2 strains. CWD has also been transmitted, albeit with varying efficiency, to Tg mice expressing mouse PrP.^27,52 In the former study, a single mule deer isolate produced disease in all inoculated Tga20 mice. On successive passages, incubation times dropped to ∼160 d. In the second study, one elk isolate from a total of eight deer and elk CWD isolates induced disease in 75% of inoculated Tg4053 mice. It is worth noting that the distribution of lesions in both studies appeared to resemble the CWD1 pattern. Low efficiency CWD prion transmission was also recorded in hamsters and Tg mice expressing Syrian hamster PrP.³¹ In that study, during serial passage of mule deer CWD, fast and slow incubation time strains with different patterns of brain pathology and PrP^Sc deposition were also isolated.

Finally, our identification and characterization of distinct CWD strains and the influence of PrP primary structure on their stabilities, is of importance when considering the potential for transmission to species outside the family Cervidae. The appearance of variant Creutzfeldt-Jakob disease (vCJD) following human exposure to bovine spongiform encephalopathy (BSE),^53,54 our demonstration of CWD prions in muscle55 and antler velvet,³⁰ as well as Race and colleagues' description of CWD prions in deer fat,⁵⁶ place the human species barrier to CWD at the forefront of public health concerns. North American hunters harvest thousands of deer and elk each year. Since it is not currently mandatory to have these animals tested for CWD, it is likely that humans consume CWD prions. While CWD prions have hitherto reassuringly failed to induce disease in Tg mice expressing human PrP,^27,28,57 systematically addressing the zoonotic potential, as well as the tissue distributions of CWD1 and CWD2 strains in infected deer and elk, would nonetheless appear to be high priorities.