Journal of Molecular Biology
Volume 413, Issue 3, 28 October 2011, Pages 527-542
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Conformational Transformation and Selection of Synthetic Prion Strains

https://doi.org/10.1016/j.jmb.2011.07.021Get rights and content

Abstract

Prion protein is capable of folding into multiple self-replicating prion strains that produce phenotypically distinct neurological disorders. Although prion strains often breed true upon passage, they can also transform or “mutate” despite being devoid of nucleic acids. To dissect the mechanism of prion strain transformation, we studied the physicochemical evolution of a mouse synthetic prion (MoSP) strain, MoSP1, after repeated passage in mice and cultured cells. We show that MoSP1 gradually adopted shorter incubation times and lower conformational stabilities. These changes were accompanied by structural transformation, as indicated by a shift in the molecular mass of the protease-resistant core of MoSP1 from approximately 19 kDa [MoSP1(2)] to 21 kDa [MoSP1(1)]. We show that MoSP1(1) and MoSP1(2) can breed with fidelity when cloned in cells; however, when present as a mixture, MoSP1(1) preferentially proliferated, leading to the disappearance of MoSP1(2). In culture, the rate of this transformation process can be influenced by the composition of the culture media and the presence of polyamidoamines. Our findings demonstrate that prions can exist as a conformationally diverse population of strains, each capable of replicating with high fidelity. Rare conformational conversion, followed by competitive selection among the resulting pool of conformers, provides a mechanism for the adaptation of the prion population to its host environment.

Introduction

In prion diseases, including Creutzfeldt–Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle, an alternatively folded conformer of prion protein (PrP) propagates by catalyzing a posttranslational structural transition, utilizing endogenous cellular PrP (PrPC) as substrate.1, 2 This transition converts the α-helix-rich PrPC into pathogenic, aggregation-prone, β-sheet-rich conformers, termed PrPSc.3, 4, 5 This conversion can occur spontaneously or be induced by autosomal dominant mutations in the PrP gene PRNP.6, 7 Alternatively, prion disease can result from exposure to exogenous PrPSc.5

The pioneering work of Pattison and Jones,8 Dickinson and Meikle,9 and Bruce and Dickinson10 indicated that the sheep scrapie agent could propagate as multiple phenotypically distinct strains. More recent work has identified the existence of strains within prions derived from other species.11, 12 Prion strains can be distinguished based on their pathogenic properties (incubation times, neuropathology, and ability to infect cell lines) and biochemical characteristics (electrophoretic mobility, glycoform ratio, conformational stability, exposure of antibody epitopes, and affinity for conjugated polymers).13, 14, 15, 16 Evidence suggests that these strain-specific properties are enciphered in the conformation of PrPSc.11, 14, 17 The unique biological and biochemical signatures of strains can propagate faithfully in animals, cell culture, and in vitro amplification experiments.5, 12, 18, 19, 20

Although natural prion strains can breed true upon serial passage, several observations suggest that they are capable of altering their molecular properties in order to adapt to new hosts and environments, in a process referred to as “strain adaptation.” This phenomenon is frequently observed when prions are transmitted to a different host species. The interspecies transmission of prions is typically an inefficient process characterized by long incubation times and low infection rates.21, 22, 23, 24 Evidence suggests that this species barrier is a result of incompatibilities between the conformation of the infecting prion strain and the conformation of the host PrPC, due in part to differences in amino acid sequences.23, 25 However, upon repeated passage, the prion conformation changes in its new host, and the incubation period gradually shortens.23

Prion adaptation has also been observed in the form of dynamic interconversions of strains derived from the same organism. Passaging of biologically cloned transmissible mink encephalopathy (TME) prions into Syrian golden hamsters can result in two phenotypically distinct strains: a long-incubation-period strain called drowsy (DY) and a short-incubation-period strain named hyper (HY).26 Upon initial infection of hamsters with biologically cloned TME prions, the DY strain predominated. However, continuous serial passage of DY prions in hamsters resulted in the gradual selection of the HY strain.20, 27, 28

A similar phenomenon was observed in mice infected with variant CJD strains. Transgenic (Tg) mice expressing chimeric human/mouse PrP inoculated with variant CJD prions can harbor two distinct strains of prions.29 In mice that expressed a mixture of the two strains, the faster-replicating strain became dominant on subsequent passage.30 More recent experiments have shown that prions can also evolve in response to selective environmental pressures. The presence of the antiprion drug quinacrine31 or the glycoside hydrolase inhibitor swainsonine32 results in the selection of prion conformations that are resistant to the respective drug's actions in cell culture models. Although these observations suggest that prion strains are dynamic pathogens capable of adaptation to novel environments, the mechanism of prion strain evolution remains unknown and subject to speculation.33

Prion strain diversity has recently been augmented by the creation of infectious synthetic prions formed exclusively from bacterially derived recombinant PrP. The first synthetic prion strain, termed mouse synthetic prion (MoSP) 1, was created by refolding recombinant truncated mouse PrP(Δ23–88) into β-sheet-rich amyloid fibrils, followed by intracerebral inoculation into Tg mice expressing the same truncated PrP sequence (Tg9949).34 Although aged Tg9949 mice are prone to neurologic disease, they do not spontaneously accumulate infectious prions.35 However, when inoculated with MoSP1 fibrils, Tg9949 mice amassed protease-resistant infectious prions (rPrPSc).34 Passage of MoSP1 in wild-type (wt) inbred FVB or Tg mice overexpressing full-length PrP (Tg4053) led to diverse incubation times, suggesting that MoSP1 may be composed of multiple strains.36, 37 Further inoculations of wt and Tg mice with various PrP amyloid preparations led to the generation of additional distinct synthetic prion strains.35, 38, 39

Compared to prions that are endemic in natural populations (such as scrapie), newly created synthetic prion strains have not been exposed to extended periods of natural selection. We therefore reasoned that synthetic strains reflect unoptimized prion states that may be prone to conformational transformation and selection upon repeated passage. We serially passaged MoSP1 in mice and cultured cell lines, and monitored changes in its biochemical and biological properties. Our data indicate that MoSP1 gradually adopted shorter incubation periods and a defined set of biochemical and neuropathological properties. We demonstrate that the observed MoSP1 transformation resulted from rare conversion events, followed by competition and selection among a heterogeneous pool of prions.

Section snippets

Generation and serial passage of MoSP1 in mice

Previously, we demonstrated that inoculation of β-sheet-rich amyloid fibrils prepared from truncated (residues 89–230) recombinant mouse PrP (recMoPrP) into Tg mice expressing a similarly truncated construct (Tg9949 mice40) led to the generation of synthetic prions.34 Although Tg9949 mice inoculated with phosphate-buffered saline (PBS) or bovine serum albumin developed late-onset ataxia after 600 days, neither infectious prions nor related neuropathology was detectable in their brains.38

Discussion

Our studies demonstrate that MoSP1 undergoes profound conformational transformations during serial passage in both mice and cultured cells. These conformational changes were accompanied by marked decreases in incubation periods and changes in neuropathological profiles. Importantly, we were able to infect N2a cells with MoSP1 prions and to recapitulate their conformational transformation in culture. Recent experiments have shown that mammalian31, 32 and yeast48, 49 prions can evolve

Ethics statement

All animal experiments followed “The Guide for the Care and Use of Laboratory Animals” published by the National Research Council in 1996. All operations and procedures were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco.

Production of recMoPrP(89–230) and amyloid fibers

The expression and purification of recMoPrP(89–230) and the formation of amyloid fibers have been previously described.61 The molecular mass and purity of recMoPrP(89–230) were confirmed by laser desorption and

Acknowledgements

This work was supported by grants from the National Institutes of Health (AG02132, AG10770, AG021601, and AG031220), as well as by gifts from the G. Harold and Leila Y. Mathers Charitable Foundation, the Sherman Fairchild Foundation, and Robert Galvin. The authors thank Dr. Jan Langeveld for providing the 12B2 antibody.

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