Molecular dynamics simulations of early steps in RNA‐mediated conversion of prions

The rate‐limiting step in prion diseases is the initial transition of a prion protein from its native form into a mis‐folded state in which the protein not only forms cell‐toxic aggregates but also becomes infectious. Recent experiments implicate polyadenosine RNA as a possible agent for generating the initial seed. In order to understand the mechanism of RNA‐mediated mis‐folding and aggregation of prions, we dock polyadenosine RNA to mouse and human prion models. Changes in stability and secondary structure of the prions upon binding to polyadenosine RNA are evaluated by comparing molecular dynamics simulations of these complexes with that of the unbound prions.     

The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.     

Techniques to elucidate the conformation of prions

Proteinaceous infectious particles(prions) are unique pathogens as they are devoid of any coding nucleic acid.Whilst it is assumed that prion disease is transmitted by a misfolded isoform of the cellular prion protein, the structural insight of prions is still vague and research for high resolution structural information of prions is still ongoing. In this review, techniques that may contribute to the clarification of the conformation of prions are presented and discussed.     

A non-Q/N-rich prion domain of a foreign prion, [Het-s], can propagate as a prion in yeast

Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrP(Sc), causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine (Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions, transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN(+)] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. Taken together, these results demonstrate prion propagation in a truly foreign host. Since yeast can host non-Q/N-rich prions, such native yeast prions may exist.     

Genetics of prion infections

Although the infectious prions causing scrapie and several human transmissible neurodegenerative diseases resemble viruses in many respects, molecular and genetic analyses indicate that prions are fundamentally different from viruses in their structure and the mechanisms by which they cause disease. The only macromolecule that has been identified in infectious prion preparations is a disease-specific isoform of the prion protein, which is encoded by a host gene. A growing body of data supports the contention that prion infections represent a novel host-pathogen interaction.     

Prions in yeast

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the "protein only" model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions.     

Molecular basis of cerebral neurodegeneration in prion diseases

The biochemical nature and the replication of infectious prions have been intensively studied in recent years. Much less is known about the cellular events underlying neuronal dysfunction and cell death. As the cellular function of the normal cellular isoform of prion protein is not exactly known, the impact of gain of toxic function or loss of function, or a combination of both, in prion pathology is still controversial. There is increasing evidence that the normal cellular isoform of the prion protein is a key mediator in prion pathology. Transgenic models were instrumental in dissecting propagation of prions, disease-associated isoforms of prion protein and amyloid production, and induction of neurodegeneration. Four experimental avenues will be discussed here which address scenarios of inappropriate trafficking, folding, or targeting of the prion protein.     

Cells and prions: A license to replicate

Prion diseases are neurodegenerative, infectious disorders characterized by the aggregation of a misfolded isoform of the cellular prion protein (PrP^C). The infectious agent - termed prion - is mainly composed of misfolded PrP^Sc. In addition to the central nervous system prions can colonize secondary lymphoid organs and inflammatory foci. Follicular dendritic cells are important extraneural sites of prion replication. However, recent data point to a broader range of cell types that can replicate prions. Here, we review the state of the art in regards to peripheral prion replication, neuroinvasion and the determinants of prion replication competence.     

Antibodies to the scrapie protein decorate prion rods

Scrapie is a degenerative, transmissible neurologic disease of sheep and goats which occurs in the absence of any detectable host immune response. Antibodies to the scrapie agent have been produced after immunization of rabbits with either scrapie prions or the prion protein, PrP 27-30, purified from infected hamster brain. Immunoreactivity of the antisera was assessed by dot and Western immunoblots with purified prions and PrP 27-30. Antibodies raised against infectious prions were more immunoreactive with native than denatured preparations, whereas those raised against PrP 27-30 were more reactive with denatured prion preparations. As determined by second antibody-colloidal gold, both antisera were found to decorate scrapie prion rods in purified preparations. Antibodies to cellular filamentous proteins failed to react with PrP 27-30 or the scrapie prion rods; conversely, antibodies to PrP 27-30 did not exhibit immunoreactivity with cellular filamentous proteins. The monospecificity of the rabbit antiserum raised against PrP 27-30 was established by its reactivity after affinity purification. The purified antibodies reacted with PrP 27-30 on Western blots and with the prion rods. Considerable evidence indicates that the scrapie rods are aggregates of infectious prions; the findings presented here provide an immunologic demonstration that PrP 27-30 is a structural component of the prion rods.     

Protein misfolding cyclic amplification of infectious prions

Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP(C) and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP(Sc) and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.     

Detection of infectious prions in urine

Prions are the infectious agents responsible for prion diseases, which appear to be composed exclusively by the misfolded prion protein (PrP(Sc)). The mechanism of prion transmission is unknown. In this study, we attempted to detect prions in urine of experimentally infected animals. PrP(Sc) was detected in approximately 80% of the animals studied, whereas no false positives were observed among the control animals. Semi-quantitative calculations suggest that PrP(Sc) concentration in urine is around 10-fold lower than in blood. Interestingly, PrP(Sc) present in urine maintains its infectious properties. Our data indicate that low quantities of infectious prions are excreted in the urine. These findings suggest that urine is a possible source of prion transmission.     

In vitro generation of high-titer prions

Prions are composed mainly, if not entirely, of PrP(Sc), an infectious misfolded isoform of PrP(C), the normal isoform of the prion protein. Here we show that protein misfolding cyclic amplification (PMCA)-generated hypertransmissible mink encephalopathy (HY TME) PrP(Sc) is highly infectious and has a titer that is similar, if not identical, to that associated with brain tissue from animals infected with the HY TME agent that are in the terminal stage of disease. These data demonstrate that PMCA efficiently replicates the prion agent and provide further support for the hypothesis that in vitro-generated prions are bona fide and are not due to contamination.     

Selective incorporation of polyanionic molecules into hamster prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro.Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.     

Intriguing nucleic-acid-binding features of mammalian prion protein

In transmissible spongiform encephalopathies, the infectious material consists chiefly of a protein, the scrapie prion protein PrP(Sc), that carries no genetic coding material; however, prions are likely to have accomplices that chaperone their activity and promote the conversion of the cellular prion protein PrP(C) into the disease-causing isoform (PrP(Sc)). Recent studies from several laboratories indicate that PrP(C) recognizes many nucleic acids (NAs) with high affinities, and we correlate these findings with a possible pathophysiological role for this interaction. Thus, of the chaperones, NA is the most likely candidate for prions. The participation of NAs in prion propagation opens new avenues for developing new diagnostic tools and therapeutics to target prion diseases, as well as for understanding the function of PrP(C), probably as a NA chaperone.     

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.     

Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.     

Interspecies transmission of prions

Mammalian prions are infectious agents of proteinaceous nature that cause several incurable neurodegenerative diseases. Interspecies transmission of prions is usually impeded or impossible. Barriers in prion transmission are caused by small interspecies differences in the primary structure of prion proteins. The barriers can also depend on the strain (variant) of a transmitted prion. Interspecies barriers were also shown for yeast prions, which define some heritable phenotypes. Yeast prions reproduce all the main traits of prion transmission barriers observed for mammals. This allowed to show that the barrier in prion transmission can be observed even upon copolymerization of two prionogenic proteins. Available data allow elucidation of the mechanisms that impede prion transmission or make it impossible.     

Selection of ovine PrP high-producer subclones from a transfected epithelial cell line

The hallmarks of prion diseases are the conversion of the normal prion into an abnormal protease resistant isoform and its brain accumulation. Purification of the native abnormal prion isoform for biochemical and biophysical studies has been hampered by poor recovery from brain tissue. An epithelial cell transfected with the ovine VRQ allele prion, called Rov9, has been used to select prion high-producer cells by flow cytometry. The representative clone 4 described here produced 6.2 microg of cellular prion protein per mg of total protein extract, representing 8- to 10-fold the amount produced by the Rov9 parental cells. After exposure to the scrapie agent (PG128/98), clone 4 produced 2.6 microg of abnormal isoform per mg of total protein. When infected clone 4 cell cultures were treated with tunicamycin, 80% of the abnormal isoform was deglycosylated. The infectivity of the prions produced in clone 4 cultures was confirmed in a mouse bioassay. Such high-producer clones represent new tools for producing large amounts of glycosylated and/or non-glycosylated PrP(Sc) and for a powerful screening of clinical samples' infectivity.     

Hypothèse : le modèle du lieu de rencontre pour la maladie des prions

Prions are responsible for spongiform diseases such as scrapie and bovine spongiform encephalopathy. It is now generally accepted that the disease mechanism involves the conversion from the normal form, PrP^C, to the pathogenic form, PrP^Sc, and that this isoform is infectious. In the case of scrapie, 15 different forms of the disease have been described and some of these different phenotypes can be conferred by infectious prions that are themselves encoded by normal genes. We propose here that a prion with an altered structure has a correspondingly altered preference for lipids; this altered preference creates a proteolipid domain containing different lipids and other factors such as chaperonins and enzymes responsible for post-translational modifications. Normal prions associated with this abnormal domain adopt the conformation dictated by its lipidic composition (and by the other factors present) and so acquire the lipidic preference of the original pathogenic prions. These transformed prions could then create new proteolipid domains. This process may be considered as semi-conservative replication in which prion and lipids are analogous to the Watson and Crick strands and the proteolipid domain to the double helix itself.     

Intra-host mathematical model of chronic wasting disease dynamics in deer (Odocoileus)

Bioassays of native cervid hosts have established the presence of infectious chronic wasting disease (CWD) prions in saliva, blood, urine, and feces of clinically diseased and pre-clinical infected deer. The intra-host trafficking of prions from the time of initial infection to shedding has been less well defined. We created a discrete-time compartmentalized model to simulate the misfolding catalysis, trafficking, and shedding of infectious prions throughout the organ systems of CWD-infected cervids. Using parameter values derived from experimental infections of North American deer (Odocoileus spp.), the exponential-based model predicts prion deposition over time with: 1) nervous tissues containing the highest deposition of prions at 20 months post-infection and 2) excreted fluids containing low levels of prions throughout infection with the highest numbers of prions predicted to be shed in saliva and feces (as high as 10 lethal doses (1.34 × 10²⁹ prions) in 11–15 months). These findings are comparable to prion deposition described in literature as assayed by conventional and ultrasensitive amplification assays. The comparison of our model to published data suggests that highly sensitive assays (sPMCA, RT-QuIC, and bioassay) are appropriate for early prion detection in bodily fluids and secretions. The model provides a view of intra-host prion catalysis leading to pre-clinical shedding and provides a framework for continued development of antemortem diagnostic methods.