The many shades of prion strain adaptation

Abstract

In several recent studies transmissible prion disease was induced in animals by inoculation with recombinant prion protein amyloid fibrils produced in vitro. Serial transmission of amyloid fibrils gave rise to a new class of prion strains of synthetic origin. Gradual transformation of disease phenotypes and PrP^Sc properties was observed during serial transmission of synthetic prions, a process that resembled the phenomenon of prion strain adaptation. The current article discusses the remarkable parallels between phenomena of prion strain adaptation that accompanies cross-species transmission and the evolution of synthetic prions occurring within the same host. Two alternative mechanisms underlying prion strain adaptation and synthetic strain evolution are discussed. The current article highlights the complexity of the prion transmission barrier and strain adaptation and proposes that the phenomenon of prion adaptation is more common than previously thought.

When prions are transmitted between species, the transmission success is often significantly lower than serial transmission within the same host due to a species barrier.¹ Species barrier manifestations are a low attack rate and long incubation time to clinical disease or lack of clinical disease in the initial passages.^2,3 To a large extent, the species barrier is attributed to differences in amino acid sequence between host PrP^C and donor PrP^{Sc 4–6} and has also been referred to as a ‘sequence barrier’⁷. However, even for strains originating from the same species, the extent of a barrier largely depends on the strain-specific conformation of PrP^Sc. As such, it would be more appropriate to refer to the “species barrier” as a “structure-sequence barrier.” While “structure-sequence barrier” explains why some strain-specific structures are less compatible with the PrP^C sequence of a new host than others, this concept cannot account for some puzzling observations. For instance, transmission of variant Creutzfeld-Jakob disease prions from a human to transgenic mice that express human PrP^C exhibited a significant barrier, whereas transmission of the same prions to wild type mice showed little barrier if any.^8,9 It is tempting to speculate that the barrier between two species, both of which express PrP^C of identical amino acid sequences, could be due to species-specific cellular cofactors and/or species-specific PrP^C glycosyl composition. Indeed, in resent studies, cellular co-factors and, in particular, lipids were found to be involved in maintaining infectious prion conformations and controlling strain-specific features.^10,11 If the species barrier, at least under some circumstances, is attributable to changes in species-specific cofactors or differences in cellular environment, it should be referred to as a “cofactor barrier.”

Regardless of the molecular mechanisms underlying species barriers, prions are able to gradually adapt to a new host when serially transmitted within the same species following cross-species transmission. The most widely acknowledged manifestations of strain adaptation are an increase in attack rate and a decrease in incubation time to disease. A phenomenon similar to prion strain adaptation was also described in studies where transmissible prion diseases were initiated in animals by inoculating amyloid fibrils generated in vitro from recombinant PrP^12-15 (Table 1). In several independent studies conducted on mice or Syrian hamsters, rPrP fibrils gave rise to prions strains that will be referred to as strains of synthetic origin.^14,16-20 Despite the fact that synthetic prions were transmitted within the same species, multiple serial passages were required to stabilize the disease phenotypes.^15,17,21,22 In a manner similar to adaptation of prions of natural origin, the attack rate increased while the incubation time to disease shortened in the course of serial transmission of synthetic prions.^{12-15,17,19,21,23} In addition, physical properties of PrP^Sc such as conformational stability as well as neuropathological features including neurotropism and PrP^Sc deposition patterns underwent gradual transformation during serial transmission.^{12-15,17,21,23} Moreover, during the initial passages of synthetic prions a dissociation between accumulation of PK-resistant forms of PrP^Sc and progression of clinical disease was observed.^18,21 Previously dissociation between PrP^Sc and clinical disease was found to accompany cross-species prion transmission, for example, transmission of BSE to mice.²⁴

Table 1. Common features observed during serial transmission of strains of synthetic origin and cross-species transmission of strain of natural origin.

	Cross-species transmission	Transmission of synthetic strains
Shortening of the incubation time	3, 45–48	12-15, 17, 19, 21, 23
Change in neurotropism and PrPSc deposition pattern	49	21
Change in PrPSc physical properties	50	12–15, 17, 23
Dissociation between clinical disease and accumulation of PK-resistant PrPSc	24	18, 21
Strain breakdown	45, 46, 51, 52	21

The aforementioned observations are typical manifestation of prion strain adaptation to a new host (Table 1). These similarities suggest that the mechanisms that account for adaptation of strains of natural origin to a new host and evolution of strains of synthetic origin might be the same or very similar. Because serial transmissions of synthetic prions were conducted within the same host, long incubation time to disease and low initial attack rate could not be attributed to a “sequence barrier.” Recent studies revealed that PrP folding patterns within amyloid fibrils produced in vitro using rPrP were significantly different from patterns that PrP adapt within PrP^Sc.^25,26 These structural differences could be due to lack of posttranslational modifications in rPrP found in PrP^C. It is likely that posttranslational modifications limit the subset of self-replicating states accessible to PrP^C creating a barrier for recruiting and converting PrP^C.²⁷ If this is true, synthetic strain adaptation should be attributable to transformation of rPrP amyloid structures to PrP^Sc-like structures accessible for PrP^C. In addition, synthetic strain adaptation could also be attributed to evolution of co-factor dependency, i.e., a transformation of co-factor independent rPrP amyloid structures to PrP^Sc-like structures that rely on cellular co-factors.

While the evolution of synthetic strains exhibits remarkable parallels with prion strain adaptation, there are some differences between the two processes. For instance, following cross-species intracerebral inoculations prions replicate more effectively in lymphoid tissues than in brain.²⁸ In opposition to the high permissiveness of lymphoid tissues to cross-species prion invasion, lymphotropism of synthetic strains appears to emerge slowly or at later stages, i.e., after more than one serial passages.²¹ However, the evolution of synthetic strain lymphotropism has not yet been fully explored, and the initial lack of lymphotropism could be attributed to intracerebral inoculation route.

What are the molecular mechanisms behind natural and synthetic strain adaptation? According to the “cloud” hypothesis, pools of PrP^Sc within individual strains are intrinsically heterogeneous.²⁹ The heterogeneity presumably arises due to spontaneous ‘mutations’ or variations in PrP^Sc structure.^29,30 Upon cross-species transmission, minor PrP^Sc variants that are the most compatible with the amino acid sequence of the new host PrP^C and best fitted to replicate in a new environment receive selective advantage. An alternative to the “cloud” hypothesis is a deformed templating model. Contrary to “cloud” hypothesis, which proposes that “new” variants are selected from pre-existing cloud of variants, deformed templating model postulates that changes in replication environment including cross-species transmission play an active role in generating new PrP^Sc variants ^31,32. According to this model, under circumstances where the strain-specific PrP^Sc template is not compatible with the new replication environment or the amino acid sequence of a new host PrP^C, a range of new PrP^Sc variants are generated via deformed templating with the help of PrP^Sc seeds. While the majority of newly generated variants might not be effective in replicating either, a variant that fits well to the environment or is compatible with the PrP^C sequence of the new host will eventually emerge through multiple trial and error seeding events. Both models, however, propose that changes in replication environment are important for selecting the variants (either pre-existing or generated de novo) that fit the best to the new environment.^31,32

The “cloud” hypothesis does not clearly specify the origin of minor PrP^Sc variants and assumes that structural variations might arise spontaneously. In contrast to the “cloud” hypothesis, deformed templating proposes that changes in the replication environment play an active role in boosting conformational diversity of the PrP^Sc pool. In direct support of the deformed templating model, experiments on fibrillation of mouse rPrP seeded with amyloid fibrils made using hamster rPrP revealed a switching in the PrP folding pattern within individual fibrils.³³ The change in folding pattern occurred only in those cross-seeded reactions, in which the folding pattern of hamster seeds was not compatible with the amino acid sequence of a mouse substrate.^33,34 Remarkably, the hybrid fibrils produced as a result of cross-seeding were composed of hamster and mouse rPrP with two distinct cross-β structures.^26,33 Other studies demonstrated that changes in prion replication environment and, specifically, adaptation of hamster strains 263K and Hyper to RNA-depleted brain homogenate and then re-adaptation to brain homogenate containing RNA in PMCA resulted in stable transformation of PrP^Sc physical properties.³⁵ Remarkably, the newly emerged PrP^Sc variants were not present in the original 263K or Hyper brain-derived materials, but emerged de novo as a result of changes in replication environment. These studies provided a direct illustration that a change in replication environment, whether it is a change in the amino acid sequence of a substrate or in the biochemical environment, resulted in an emergence of new self-replicating states that were absent in the original pools of seeding material.

The remarkable ability of prions to adapt to new cellular environments is highlighted in recent studies on drug resistance. In the presence of carefully selected replication inhibitors, prions were found to gradually acquire drug resistance.^30,36-39 Currently, it is unclear whether drug resistance develops as a result of selective amplification of pre-existing minor variants as proposed by the ‘cloud’ hypothesis, or drug-resistant PrP^Sc variants evolved de novo as postulated by deformed templating mechanism. While prions can acquire resistance to a diverse range of compounds, it is difficult to imagine that conformational heterogeneity of a “cloud” is boundless, i.e., a “cloud” consists of unlimited number of minor variants that fit well to replicate in any and every circumstance including diverse cellular environments or in the presence of chemical inhibitors. The mechanism where a novel PrP^Sc conformation can emerge de novo helps to explain the remarkable potential of prions for adapting to diverse new environments.

The “cloud” and deformed templating hypotheses are not mutually exclusive. The deformed templating does not exclude structural heterogeneity of PrP^Sc pools of natural or synthetic origin, but provides a mechanism by which conformational members of a “cloud” undergo structural alterations in a new environment and give a rise to a new “cloud.” It is not entirely clear what are the driving forces behind diversifying events and the frequency or rate of diversifying events under constant replication environment. The “cloud” hypothesis assumes that conformational diversity of a cloud is due to spontaneous ‘mutations’ that occur continuously at low rates.

When amplified in cultured cells or using different PMCA formats, prion properties were found to undergo gradual changes in a manner that appears similar to prion adaptation.^30,36,40-42 For example, adding the glycosylation inhibitor swainsonin to cultured cells led to the gradual transformation of prion populations.^30,42,43 In the course of serial amplification in PMCA, the strain-specific amplification rate increased gradually,⁴¹ and strain-specific secondary structure monitored by FTIR also underwent gradual changes.⁴⁴ If partially deglycosylated PrP^C was used as a substrate in PMCA, strain-specific proteinase K resistance profiles showed significant transformation.⁴⁰ However, when tested in animal bioassay the changes in PrP^Sc properties acquired upon replication in PMCA or in cultured cells were often found to be reversible and, most importantly, did not lead to stable changes in disease phenotype.^40-43 While PrP^Sc transformations in diverse cellular or biochemical environments are indicative of the dynamic nature of prions, they should not be confused with actual strain adaptation. Instead, it is worthwhile to compare this phenomenon to a norm of reaction, a concept that describes phenotypic variations of a single genotype across a range of environments. The concepts of norm of reaction and phenotypic plasticity were introduced into population genetics to describe variations in phenotype and the ability of an organism to change its phenotype, respectively, in response to changes in the environment. For instance, plants can acquire multiple morphologically distinct phenotypes within a single genotype to fit into a diverse range of environments. Noteworthy, phenotypic plasticity is not attributed to mutations but to an intrinsic norm of reaction. A concept analogous to norm of reaction could be useful for describing reversible changes in PrP^Sc features observed across diverse replication environments, such as different cultured cells and PMCA formats that do not lead to stable changes in disease phenotype or characteristics that define strain-ness.

Glossary

Abbreviations:

PrP^C

normal cellular isoform of the prion protein

PrP^Sc

abnormal, disease-associated isoform of the prion protein

PrP

prion protein

rPrP

recombinant PrP

PMCA

Protein misfolding cyclic amplification

Makarava N, Kovacs GG, Savtchenko R, Alexeeva I, Budka H, Rohwer RG, Baskakov IV. Stabilization of a prion strain of synthetic origin requires multiple serial passages. J Biol Chem. 2012;287:30205–14. doi: 10.1074/jbc.M112.392985.

Published Online: 02/11/2014

10.4161/pri.27836