Highly Infectious Prions Generated by a Single Round of Microplate-Based Protein Misfolding Cyclic Amplification

Moudjou, Mohammed; Sibille, Pierre; Fichet, Guillaume; Reine, Fabienne; Chapuis, Jérôme; Herzog, Laëtitia; Jaumain, Emilie; Laferrière, Florent; Richard, Charles-Adrien; Laude, Hubert; Andréoletti, Olivier; Rézaei, Human; Béringue, Vincent

doi:10.1128/mbio.00829-13

Cited by 49 publications

(92 citation statements)

References 67 publications

(102 reference statements)

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“…Amplifications were performed according to the miniaturized bead PMCA assay (48). To test the conversion of deletion mutants in vitro, we used cell lysates to which 5% brain homogenate from PrP null mouse was added as the substrates.…”

Section: Methodsmentioning

confidence: 99%

Generating Bona Fide Mammalian Prions with Internal Deletions

Muñoz-Montesino

Sizun

Moudjou

et al. 2016

J Virol

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Mammalian prions are PrP proteins with altered structures causing transmissible fatal neurodegenerative diseases. They are self-perpetuating through formation of beta-sheet-rich assemblies that seed conformational change of cellular PrP. Pathological PrP usually forms an insoluble protease-resistant core exhibiting beta-sheet structures but no more alpha-helical content, loosing the three alpha-helices contained in the correctly folded PrP. The lack of a high-resolution prion structure makes it difficult to understand the dynamics of conversion and to identify elements of the protein involved in this process. To determine whether completeness of residues within the protease-resistant domain is required for prions, we performed serial deletions in the helix H2 C terminus of ovine PrP, since this region has previously shown some tolerance to sequence changes without preventing prion replication. Deletions of either four or five residues essentially preserved the overall PrP structure and mutant PrP expressed in RK13 cells were efficiently converted into bona fide prions upon challenge by three different prion strains. Remarkably, deletions in PrP facilitated the replication of two strains that otherwise do not replicate in this cellular context. Prions with internal deletion were self-propagating and de novo infectious for naive homologous and wild-type PrP-expressing cells. Moreover, they caused transmissible spongiform encephalopathies in mice, with similar biochemical signatures and neuropathologies other than the original strains. Prion convertibility and transfer of strain-specific information are thus preserved despite shortening of an alpha-helix in PrP and removal of residues within prions. These findings provide new insights into sequence/structure/infectivity relationship for prions. IMPORTANCEPrions are misfolded PrP proteins that convert the normal protein into a replicate of their own abnormal form. They are responsible for invariably fatal neurodegenerative disorders. Other aggregation-prone proteins appear to have a prion-like mode of expansion in brains, such as in Alzheimer's or Parkinson's diseases. To date, the resolution of prion structure remains elusive. Thus, to genetically define the landscape of regions critical for prion conversion, we tested the effect of short deletions. We found that, surprisingly, removal of a portion of PrP, the C terminus of alpha-helix H2, did not hamper prion formation but generated infectious agents with an internal deletion that showed characteristics essentially similar to those of original infecting strains. Thus, we demonstrate that completeness of the residues inside prions is not necessary for maintaining infectivity and the main strain-specific information, while reporting one of the few if not the only bona fide prions with an internal deletion.

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Section: Methodsmentioning

confidence: 99%

Generating Bona Fide Mammalian Prions with Internal Deletions

Muñoz-Montesino

Sizun

Moudjou

et al. 2016

J Virol

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“…La première étape est supposée favoriser la conversion de la PrP C en PrP Sc et sa polymérisation, la seconde, fragmenter les assemblages et générer ainsi de nouvelles sous-unités polymé-risantes pour un second cycle. Il en résulte une amplification exponentielle des molécules de PrP Sc initialement présentes et de l'infectiosité associée (Moudjou et al, 2014). Les prions générés conservent généralement les mêmes propriétés de souche que le prion introduit dans la réaction (Castilla et al, 2008).…”

Section: Risques Associés Aux Maladies à Prions Animalesunclassified

Les prions, agents d’encéphalopathies transmissibles chez les mammifères

Béringue¹

2017

bavf

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Les maladies à prion sont des affections neurodégénératives d'issue fatale touchant principalement les ruminants d'élevage et l'homme. Les prions sont des agents infectieux constitués d'une protéine de l'hôte, la PrP qui, sous sa forme pathologique, s'agrège en polymères de type amyloïde autopropagés. Bien que totalement dépourvus de génome, les prions montrent des propriétés analogues à celles d'autres agents infectieux, dont la diversité de souche, le tropisme tissulaire et la capacité à franchir les barrières de transmission interspécifique. Le but de cette revue est de faire une synthèse des connaissances et des avancées récentes dans ce domaine complexe.

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“…The sensitivity achieved is generally superior to that of the bioassay by two to three orders of magnitude (Saa et al, 2006;Johnson et al, 2012;Makarava et al, 2012;Moudjou et al, 2013).…”

Section: Correlation Of Sensitivity Between In Vitro Amplification Anmentioning

confidence: 99%

“…A number of studies have directly compared the performances of cell-free and animal bioassays in determining endpoint titrations (Saa et al, 2006;Wilham et al, 2010;Johnson et al, 2012;Makarava et al, 2012;Moudjou et al, 2013). The sensitivity achieved is generally superior to that of the bioassay by two to three orders of magnitude (Saa et al, 2006;Johnson et al, 2012;Makarava et al, 2012;Moudjou et al, 2013).…”

Section: Correlation Of Sensitivity Between In Vitro Amplification Anmentioning

confidence: 99%

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Protocol for further laboratory investigations into the distribution of infectivity of Atypical BSE

2014

EFSA Journal

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Information on the pathogenesis and tissue distribution of Atypical Bovine Spongiform Encephalopathy (BSE) in cattle through the study of field cases and experimental transmission studies is lacking. The latter are limited to transmission of Atypical BSE through intracerebral (i.c.) inoculation of cattle. All data currently available relate to the presence or absence of PrP Sc , but do not quantify relative amounts of PrP Sc or levels of infectivity. A laboratory protocol for further studies is recommended, to allow the assessment of the relative infectious titre, PrP Sc accumulation and prion seeding activity in the tissues of cattle that developed H-BSE or L-BSE (using posterior brainstem as a reference). Tissues to be covered by those studies are categorised in three priorities, based on their inclusion in the list of specific risk material in cattle, on the presence of infectivity, or PrP Sc presence, demonstrated in Atypical BSEs or other Transmissible Spongiform Encephalopathies (TSEs) in ruminants, and on the importance in terms of input into the food chain in the EU. The protocol provides details in terms of the minimum number of animals to be tested, processing and preparation of tissues, and methods to be used to identify abnormal PrP and quantify infectivity, also depending on the expected level of infectivity and amount of tissue available for analysis. It is recommended that, through the implementation of the protocol, information should also be obtained on the performance of currently validated rapid tests for TSE active surveillance in cattle/bioassay for detecting H-BSE and L-BSE agents.

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Highly Infectious Prions Generated by a Single Round of Microplate-Based Protein Misfolding Cyclic Amplification

Cited by 49 publications

References 67 publications

Generating Bona Fide Mammalian Prions with Internal Deletions

Generating Bona Fide Mammalian Prions with Internal Deletions

Les prions, agents d’encéphalopathies transmissibles chez les mammifères

Protocol for further laboratory investigations into the distribution of infectivity of Atypical BSE

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