Mutation and Selection of Prions

The author has declared that no competing interests exist. 
 
 
 
This study was supported by grants from the National Institutes of Health (1RO1NSO59543, 1R01NSO67214) and the Alafi Family Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Replication of Prions
Prions consist mainly, if not solely, of PrP Sc (scrapie prion protein), aggregated conformers of the GPI-linked host glycoprotein PrP C (cellular prion protein). PrP Sc propagates by converting PrP C to a replica of itself ( Figure 1A). PrP C may exist as an equilibrium mixture of conformers, some of which can accrete to PrP Sc ''seeds'' at a critical rate [1,2]. This seeding model is supported by the protein misfolding cyclic amplification (PMCA) reaction, in which brain homogenate, as a source of PrP C , is spiked with a seed of infected brain homogenate and subjected to multiple cycles of sonication and incubation, ultimately yielding a vast excess of infectious prions [3]. Infectious prions arose spontaneously in PMCA-mediated, cell-free reactions from defined components [4], in particular from recombinant PrP, a phospholipid, and poly(A) or poly(dT) [5], definitively laying to rest the perennial proposal that the infectious agent is a virus-like entity [6]. Prion-like, seeded conversion into an aggregated state has been proposed for several mammalian proteins such as Abeta, a-synuclein, or serum amyloid, which underlie protein misfolding diseases, and for several fungal, in particular yeast, proteins.

Prion Strains
Prion populations may present as distinct strains: these differ in their phenotypic properties but are associated with PrP Sc having the same amino acid sequence. Murine prion strains, originally characterized by the incubation time and the neuropathology they elicit, can be propagated indefinitely in mice homozygous for the PrP gene. Many ''classical'' strains currently propagated in mice and hamsters, such as 79A, 22L, and ME7, originated from scrapie-infected sheep or goats [7] and were cloned by endpoint dilution in mice.
Strain-specific properties of the prion are believed to be enciphered in the conformation of the cognate PrP Sc [8], and indeed, distinct strains are often associated with PrP Sc species differing in physicochemical properties. Experiments with yeast prion strains have shown that specific conformations can be propagated in vitro by pure, unglycosylated proteins [9]. Nonetheless, in view of the vast multiplicity of mammalian prion strains and their tropism for particular cell lines, it is conceivable that post translational modifications of PrP, such as glycosylation or association with some cellular components, might favor certain PrP conformations and hence account for cell-specific preferential propagation of particular strains.

The Species Barrier
In general, there is a considerable barrier to transmission of prions between animal species, in that even massive intracerebral trans-species inoculation causes disease at only low frequency (low ''attack rate'') and/or only after very long incubation times, if at all. This barrier was abolished in some instances by replacing the PrP gene of the recipient by its counterpart from the donor, but clearly factors other than mismatch of PrP sequences contribute to the incompatibility. Importantly, when prions are serially transmitted from the initial trans-species recipients to further animals of the same species, attack rates increase and incubation times decrease, reflecting ''adaptation'' to the new host [10]. ''Adaptation'' implies as a first step accretion of PrP C from the recipient host to the incoming PrP Sc seed, which may be a very inefficient process if the amino acid sequence of the host PrP entrains a spectrum of conformations that are poorly compatible with that of the seed. Efficient propagation may only be enabled when the conformation of the seed changes, perhaps initially at the ''growing end'' [11], resulting in a ''mutation'' at the conformational level. Subsequently, prions may evolve to replicate more rapidly in the new host, accounting for the striking reduction of their incubation period as they are sequentially transferred within the new species.
In some instances, transfer of a prion strain from one species to another, followed by several passages in the original host species, led to emergence of mutant strains. For example, when cloned murine 139A prions were passaged through hamster and subsequently passaged repeatedly in mouse a new strain, 139A-H2M, was recovered; however, ME7 subjected to the same procedure remained apparently unchanged [12].

Evolution of Prions
The finding that many murine prion strains replicated efficiently in selected murine cell lines created important new experimental opportunities. In particular, the slow, expensive, and imprecise mouse-based bioassay for murine prions could be replaced by a humane, rapid, and precise cell-based procedure, the standard scrapie cell assay (SSCA) [13]. The differential susceptibility of cell lines to various prion strains provided the basis of the cell panel assay (CPA), which rapidly differentiates between various prion strains on the basis of their cell tropism and their susceptibility to various drugs, such as swainsonine or kifunensine [14,15].
The CPA revealed that serial propagation of brain-derived 22L prions in PK1 cells led to progressive change in their properties; while initially able to propagate in R33 cells (''R33 competent'') or in PK1 cells in the presence of swainsonine (''swainsonine resistant''), the prions gradually became completely R33 incompetent and swainsonine-sensitive ( Figure 1B). When these ''celladapted'' prions were returned to mouse brain, they gradually reacquired their former properties and became indistinguishable from the original 22L strain [16]. Along similar lines, when swainsonine-sensitive prions were propagated in PK1 cells in the presence of the drug, a swainsonine-resistant prion population emerged after a few passages, documenting adaptation to the new environment. After withdrawal of the drug, further propagation for several splits again yielded drug-sensitive prions [16]. These findings suggested that prion populations constitute so-called quasispecies [17], that is, they are composed of a variety of conformational variants, each present at a low level; when the environment changes, the most efficiently replicating variant becomes the predominant component of the population, which then constitutes a distinct sub-strain [1,16,18]. Indeed, PK1 celladapted 22L populations were found to contain about 0.5% swainsonine-resistant variants before ever being exposed to the drug [16]. Because the 22L prions used in these experiments had been cloned by endpoint dilution years earlier, heterogeneity must have arisen by a mutation-like process in the interim. Mutations in the case of prions represent conformational changes and not modifications at the level of the protein sequence, because PrP is encoded by the host genome and the mutation is inherent to the proteinaceous particle. To verify whether heterogeneity of prion populations came about by mutation, swainsonine-sensitive prions were cloned by endpoint dilution into PK1 cells, and the infected cells were propagated serially for up to 100 doublings and challenged with swainsonine to determine at which stage the prion populations acquired the capacity for becoming resistant to the drug. Early after cloning the populations were incapable of doing so, but most clones developed this capability after 31-86 doublings ( Figure 1C). However, at least one of nine populations failed to do so even after 116 doublings, suggesting that the prions were heterogeneous in regard to their ability to develop swainsonine resistance [11,16]. Acquisition of drug resistance by murine prions has also been reported by Ghaemmaghami et al. [19] and by yeast prions by Shorter [20]. Most if not all of the prion variants, or substrains, described above were reversible, suggesting that the underlying conformations were readily interconvertible. In contrast, strains are very stable, at least as long as they are propagated in the same species. As shown in Figure 2, this suggests a low activation energy barrier between sub-strains, readily surmountable under physiological conditions, while high activation energy barriers prevent conversion between strains.

Concluding Thoughts
The finding that prions can acquire resistance to drugs has significant implications for drug design. Drugs targeted to PrP Sc may have to be administered in combination, as in the case of viruses, in particular HIV. Alternatively, drugs could be targeted to bind and stabilize PrP C or, in view of the finding that ablation of PrP C , at least in animals, is not detrimental to health [21,22], to suppress its synthesis. At present no therapeutically useful drugs are available, but deepening insight into the molecular biology of prions may pave the way to novel approaches. Figure 2. Conjectural free energy landscape for prion strains and sub-strains. Sub-strains are depicted as distinguishable collectives of prions that can interconvert readily because they are separated by activation energy barriers that can be overcome in a particular environment under physiological conditions, while strains are separated by high energy barriers. The extent to which the individual wells are populated (red blocks) is determined by the accumulation rate of the particular sub-strain. When the environment changes, for example when prions are transferred between distinct tissues, different sub-strains may be favored. Adapted from reference [18]. doi:10.1371/journal.ppat.1002582.g002