28 results on '"Laude, H"'
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2. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus
- Author
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Krempl, C, primary, Schultze, B, additional, Laude, H, additional, and Herrler, G, additional
- Published
- 1997
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3. Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus
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Benbacer, L, primary, Kut, E, additional, Besnardeau, L, additional, Laude, H, additional, and Delmas, B, additional
- Published
- 1997
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4. Major receptor-binding and neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein
- Author
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Godet, M, primary, Grosclaude, J, additional, Delmas, B, additional, and Laude, H, additional
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- 1994
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5. Determinants essential for the transmissible gastroenteritis virus-receptor interaction reside within a domain of aminopeptidase-N that is distinct from the enzymatic site
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Delmas, B, primary, Gelfi, J, additional, Kut, E, additional, Sjöström, H, additional, Noren, O, additional, and Laude, H, additional
- Published
- 1994
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6. Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus
- Author
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Laude, H, primary, Gelfi, J, additional, Lavenant, L, additional, and Charley, B, additional
- Published
- 1992
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7. Assembly of coronavirus spike protein into trimers and its role in epitope expression
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Delmas, B, primary and Laude, H, additional
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- 1990
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8. Induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein E1
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Charley, B, primary and Laude, H, additional
- Published
- 1988
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9. Absence of Evidence for a Causal Link between Bovine Spongiform Encephalopathy Strain Variant L-BSE and Known Forms of Sporadic Creutzfeldt-Jakob Disease in Human PrP Transgenic Mice.
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Jaumain E, Quadrio I, Herzog L, Reine F, Rezaei H, Andréoletti O, Laude H, Perret-Liaudet A, Haïk S, and Béringue V
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- Animals, Brain metabolism, Brain pathology, Cattle, Creutzfeldt-Jakob Syndrome etiology, Creutzfeldt-Jakob Syndrome transmission, Disease Models, Animal, Encephalopathy, Bovine Spongiform etiology, Encephalopathy, Bovine Spongiform transmission, Genetic Variation, Host Specificity, Humans, Mice, Mice, Transgenic, PrPC Proteins genetics, PrPC Proteins pathogenicity, PrPSc Proteins genetics, PrPSc Proteins pathogenicity, Creutzfeldt-Jakob Syndrome genetics, Encephalopathy, Bovine Spongiform genetics
- Abstract
Prions are proteinaceous pathogens responsible for subacute spongiform encephalopathies in animals and humans. The prions responsible for bovine spongiform encephalopathy (BSE) are zoonotic agents, causing variant Creutzfeldt-Jakob disease (CJD) in humans. The transfer of prions between species is limited by a species barrier, which is thought to reflect structural incompatibilities between the host cellular prion protein (PrP
C ) and the infecting pathological PrP assemblies (PrPSc ) constituting the prion. A BSE strain variant, designated L-BSE and responsible for atypical, supposedly spontaneous forms of prion diseases in aged cattle, demonstrates zoonotic potential, as evidenced by its capacity to propagate more easily than classical BSE in transgenic mice expressing human PrPC and in nonhuman primates. In humanized mice, L-BSE propagates without any apparent species barrier and shares similar biochemical PrPSc signatures with the CJD subtype designated MM2-cortical, thus opening the possibility that certain CJD cases classified as sporadic may actually originate from L-type BSE cross-transmission. To address this issue, we compared the biological properties of L-BSE and those of a panel of CJD subtypes representative of the human prion strain diversity using standard strain-typing criteria in human PrP transgenic mice. We found no evidence that L-BSE causes a known form of sporadic CJD., Importance: Since the quasi-extinction of classical BSE, atypical BSE forms are the sole BSE variants circulating in cattle worldwide. They are observed in rare cases of old cattle, making them difficult to detect. Extrapolation of our results suggests that L-BSE may propagate in humans as an unrecognized form of CJD, and we urge both the continued utilization of precautionary measures to eliminate these agents from the human food chain and active surveillance for CJD phenotypes in the general population., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)- Published
- 2016
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10. Generating Bona Fide Mammalian Prions with Internal Deletions.
- Author
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Munoz-Montesino C, Sizun C, Moudjou M, Herzog L, Reine F, Chapuis J, Ciric D, Igel-Egalon A, Laude H, Béringue V, Rezaei H, and Dron M
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- Amino Acid Sequence, Animals, Mice, Mice, Transgenic, PrPC Proteins chemistry, Protein Conformation, Sequence Homology, Amino Acid, Sheep, Structure-Activity Relationship, Epithelial Cells metabolism, PrPC Proteins genetics, PrPC Proteins metabolism, Scrapie metabolism, Sequence Deletion
- Abstract
Unlabelled: 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., Importance: Prions 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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2016
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11. Mutated but Not Deleted Ovine PrP(C) N-Terminal Polybasic Region Strongly Interferes with Prion Propagation in Transgenic Mice.
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Khalifé M, Reine F, Paquet-Fifield S, Castille J, Herzog L, Vilotte M, Moudjou M, Moazami-Goudarzi K, Makhzami S, Passet B, Andréoletti O, Vilette D, Laude H, Béringue V, and Vilotte JL
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- Animals, Disease Models, Animal, Mice, Transgenic, Mutation, Missense, Sequence Deletion, Sheep, Mutant Proteins genetics, Mutant Proteins metabolism, PrPC Proteins genetics, PrPC Proteins metabolism, Prion Diseases pathology
- Abstract
Unlabelled: Mammalian prions are proteinaceous infectious agents composed of misfolded assemblies of the host-encoded, cellular prion protein (PrP). Physiologically, the N-terminal polybasic region of residues 23 to 31 of PrP has been shown to be involved in its endocytic trafficking and interactions with glycosaminoglycans or putative ectodomains of membrane-associated proteins. Several recent reports also describe this PrP region as important for the toxicity of mutant prion proteins and the efficiency of prion propagation, both in vitro and in vivo. The question remains as to whether the latter observations made with mouse PrP and mouse prions would be relevant to other PrP species/prion strain combinations given the dramatic impact on prion susceptibility of minimal amino acid substitutions and structural variations in PrP. Here, we report that transgenic mouse lines expressing ovine PrP with a deletion of residues 23 to 26 (KKRP) or mutated in this N-terminal region (KQHPH instead of KKRPK) exhibited a variable, strain-dependent susceptibility to prion infection with regard to the proportion of affected mice and disease tempo relative to findings in their wild-type counterparts. Deletion has no major effect on 127S scrapie prion pathogenesis, whereas mutation increased by almost 3-fold the survival time of the mice. Deletion marginally affected the incubation time of scrapie LA19K and ovine bovine spongiform encephalopathy (BSE) prions, whereas mutation caused apparent resistance to disease., Importance: Recent reports suggested that the N-terminal polybasic region of the prion protein could be a therapeutic target to prevent prion propagation or toxic signaling associated with more common neurodegenerative diseases such as Alzheimer's disease. Mutating or deleting this region in ovine PrP completes the data previously obtained with the mouse protein by identifying the key amino acid residues involved., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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12. Accelerated, spleen-based titration of variant Creutzfeldt-Jakob disease infectivity in transgenic mice expressing human prion protein with sensitivity comparable to that of survival time bioassay.
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Halliez S, Reine F, Herzog L, Jaumain E, Haïk S, Rezaei H, Vilotte JL, Laude H, and Béringue V
- Subjects
- Animals, Biological Assay, Humans, Mice, Mice, Transgenic, Sensitivity and Specificity, Time Factors, Clinical Laboratory Techniques methods, Creutzfeldt-Jakob Syndrome diagnosis, Prions analysis, Spleen chemistry
- Abstract
Unlabelled: The dietary exposure of the human population to the prions responsible for the bovine spongiform encephalopathy (BSE) epizooty has led to the emergence of variant Creutzfeldt-Jakob disease (vCJD). This fatal, untreatable neurodegenerative disorder is a growing public health concern because the prevalence of the infection seems much greater than the disease incidence and because secondary transmission of vCJD by blood transfusion or use of blood products has occurred. A current limitation in variant CJD risk assessment is the lack of quantitative information on the infectivity of contaminated tissues. To address this limitation, we tested the potential of a transgenic mouse line overexpressing human prion protein (PrP), which was previously reported to propagate vCJD prions. Endpoint titration of vCJD infectivity in different tissues was evaluated by two different methods: (i) the "classical" bioassay, based on the appearance of clinical symptoms and the detection of pathological prion protein in tissues of the inoculated mouse, and (ii) a shortened bioassay based on the detection of the protein in the mouse spleen at defined time points. The two methods proved equally sensitive in quantifying infectivity, even after very-low-dose inoculation of infected material, but the time schedule was shortened from ~2.5 years to ~1 year with the spleen bioassay. Compared to the "gold-standard" RIII model routinely used for endpoint titration of vCJD/BSE prions, either method improved the sensitivity by >2 orders of magnitude and allowed reevaluating the infectious titer of spleen from a vCJD individual at disease end stage to >1,000-fold-higher values., Importance: Here, we provide key reevaluation of the infectious titer of variant CJD brain and spleen tissues. The highly sensitive, accelerated spleen-based assay should thus constitute a key advance for variant CJD epidemiological and risk assessment purposes and should greatly facilitate future titration studies, including, for example, those aimed at validating decontamination procedures. The overlooked notion that the lymphoid tissue exhibits a higher capacity than the brain to replicate prions even after low-dose infection raises new questions about the molecular and/or cellular determinant(s) involved, a key issue regarding potent silent carriers of variant CJD in the lymphoid tissue., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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13. Prion propagation in cells expressing PrP glycosylation mutants.
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Salamat MK, Dron M, Chapuis J, Langevin C, and Laude H
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- Animals, Cell Line, Cell Membrane chemistry, Glycosylation, Golgi Apparatus chemistry, Prions genetics, Protein Transport, Rabbits, Sheep, Mutation, Missense, Prions metabolism, Protein Processing, Post-Translational
- Abstract
Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.
- Published
- 2011
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14. Infection of cell lines with experimental and natural ovine scrapie agents.
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Neale MH, Mountjoy SJ, Edwards JC, Vilette D, Laude H, Windl O, and Saunders GC
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- Animals, Female, Male, Mice, PrPC Proteins genetics, PrPC Proteins metabolism, PrPSc Proteins genetics, PrPSc Proteins pathogenicity, Scrapie genetics, Sensitivity and Specificity, Sheep, Sheep Diseases genetics, Sheep Diseases metabolism, Biological Assay methods, Cell Line, PrPSc Proteins metabolism, Scrapie metabolism
- Abstract
Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.
- Published
- 2010
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15. Prion strain- and species-dependent effects of antiprion molecules in primary neuronal cultures.
- Author
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Cronier S, Beringue V, Bellon A, Peyrin JM, and Laude H
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- Amphotericin B pharmacology, Animals, Cells, Cultured, Cricetinae, Humans, Mice, Mice, Transgenic, Neurons metabolism, Neurons pathology, PrPC Proteins drug effects, PrPC Proteins genetics, PrPC Proteins metabolism, PrPSc Proteins drug effects, PrPSc Proteins metabolism, Prion Diseases metabolism, Prion Proteins, Prions metabolism, Scrapie metabolism, Sheep, Species Specificity, Amphotericin B analogs & derivatives, Chlorpromazine pharmacology, Congo Red pharmacology, Neurons drug effects, Prions classification, Prions drug effects
- Abstract
Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents.
- Published
- 2007
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16. PrPc does not mediate internalization of PrPSc but is required at an early stage for de novo prion infection of Rov cells.
- Author
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Paquet S, Daude N, Courageot MP, Chapuis J, Laude H, and Vilette D
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- Animals, Cell Line, Epithelial Cells, Heparitin Sulfate metabolism, Mice, PrPC Proteins analysis, PrPSc Proteins analysis, PrPC Proteins metabolism, PrPSc Proteins metabolism, Scrapie metabolism
- Abstract
We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface.
- Published
- 2007
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17. Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie.
- Author
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Moudjou M, Treguer E, Rezaei H, Sabuncu E, Neuendorf E, Groschup MH, Grosclaude J, and Laude H
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- Animals, Antibodies, Monoclonal immunology, Antibody Specificity, Brain immunology, Brain metabolism, Epitopes physiology, Glycosylation, Mice, Polysaccharides immunology, PrPC Proteins genetics, PrPC Proteins immunology, Protein Isoforms chemistry, Protein Isoforms immunology, Protein Isoforms physiology, Scrapie immunology, Sheep, Epitopes chemistry, Epitopes immunology, Polysaccharides physiology, PrPC Proteins chemistry, PrPC Proteins physiology, Scrapie etiology, Scrapie metabolism
- Abstract
A key feature of prion encephalopathies is the accumulation of a misfolded form of the host glycoprotein PrP. Cell-free and cell culture studies have shown that the efficiency of conversion of PrP into the disease-associated form is influenced by its amino acid sequence and also by its carbohydrate moiety. Here, we characterize four novel glycoform-dependent monoclonal antibodies raised against prokaryotic recombinant sheep PrP. We demonstrate that these antibodies discriminate the PrP monoglycosylated species, since two of them recognize molecules that have the first Asn glycosylation site occupied (mono1) while the other two recognize molecules glycosylated at the second site (mono2). Remarkably, the recognition of PrP by the anti-mono2 antibodies was strongly influenced by the amino acid present at position 171, i.e., either Gln or Arg. This polymorphism is known to be the main determinant of susceptibility and resistance to scrapie in sheep. Altogether, our findings lead us to propose that each glycan chain controls the accessibility of PrP determinants located close upstream from their attachment site. The monoglycoform-assigned and the allotype-restricted antibodies described here, the first to date, should provide further opportunities to investigate the involvement of each glycan chain in PrP conversion in relation to prion strain diversity and the basis of the resistance conferred by the Arg-171 amino acid.
- Published
- 2004
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18. Prion infection of epithelial Rov cells is a polarized event.
- Author
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Paquet S, Sabuncu E, Delaunay JL, Laude H, and Vilette D
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- Animals, Cells, Cultured, Membrane Microdomains metabolism, Microscopy, Fluorescence, PrPC Proteins metabolism, PrPSc Proteins metabolism, PrPSc Proteins pathogenicity, Precipitin Tests, Prions metabolism, Rabbits, Scrapie metabolism, Sheep, Cell Polarity, Epithelial Cells metabolism, Prions pathogenicity
- Abstract
During prion infections, the cellular glycosylphosphatidylinositol-anchored glycoprotein PrP is converted into a conformational isoform. This abnormal conformer is thought to recruit and convert the normal cellular PrP into a likeness of itself and is proposed to be the infectious agent. We investigated the distribution of the PrP protein on the surface of Rov cells, an epithelial cell line highly permissive to prion multiplication, and we found that PrP is primarily expressed on the apical side. We further show that prion transmission to Rov cells is much more efficient if infectivity contacts the apical side, indicating that the apical and basolateral sides of Rov cells are not equally competent for prion infection and adding prions to the list of the conventional infectious agents (viruses and bacteria) that infect epithelial cells in a polarized manner. These data raise the possibility that apically expressed PrP may be involved in this polarized process of infection. This would add further support for a crucial role of PrP at the cell surface in prion infection of target cells.
- Published
- 2004
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19. Cultured peripheral neuroglial cells are highly permissive to sheep prion infection.
- Author
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Archer F, Bachelin C, Andreoletti O, Besnard N, Perrot G, Langevin C, Le Dur A, Vilette D, Baron-Van Evercooren A, Vilotte JL, and Laude H
- Subjects
- Animals, Cells, Cultured, Female, Ganglia, Spinal cytology, Mice, Mice, Transgenic, Neurons metabolism, Peripheral Nervous System metabolism, PrPC Proteins metabolism, PrPSc Proteins metabolism, Schwann Cells metabolism, Scrapie, Sheep, Neuroglia metabolism, PrPSc Proteins pathogenicity
- Abstract
Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system (CNS) by prions. Spreading of the infectious agent through the peripheral nervous system (PNS) may represent a crucial step toward CNS neuroinvasion, but the modalities of this process have yet to be clarified. Here we provide further evidence that PNS glial cells are likely targets for infection by prions. Glial cell clones originating from dorsal root ganglia of transgenic mice expressing ovine PrP (tgOv) and simian virus 40 T antigen were found to be readily infectible by sheep scrapie agent. This led us to establish two stable cell lines that exhibited features of Schwann cells. These cells were shown to sustain an efficient and stable replication of sheep prion based on the high level of accumulation of abnormal PrP and infectivity in exposed cultures. We also provide evidence for abnormal PrP deposition in peripheral neuroglial cells from scrapie-infected tgOv mice and sheep. These findings have potential implications in terms of designing new cell systems permissive to prions and of peripheral pathobiology of prion infections.
- Published
- 2004
- Full Text
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20. PrP polymorphisms tightly control sheep prion replication in cultured cells.
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Sabuncu E, Petit S, Le Dur A, Lan Lai T, Vilotte JL, Laude H, and Vilette D
- Subjects
- Animals, Cells, Cultured, Prions pathogenicity, Sheep, Alleles, Genetic Predisposition to Disease, Polymorphism, Genetic, Prions genetics, Prions metabolism, Scrapie genetics
- Abstract
Prion diseases are fatal neurodegenerative disorders of animals and humans that are characterized by the conversion of the host-encoded prion protein (PrP) to an abnormal isoform. In several species, including humans, polymorphisms in the gene encoding the PrP protein tightly control susceptibility of individuals toward this disease. In the present study we show that Rov cells expressing an ovine PrP allele ((VRQ)PrP) associated with high susceptibility of sheep to scrapie were very sensitive to sheep prion transmission and replicated the agent to high titers. In contrast, we did not find any evidence of infection when Rov cells expressed similar levels of a PrP variant ((ARR)PrP) linked to resistance. Our data provide the first direct evidence that natural PrP polymorphisms may affect prion susceptibility by controlling prion replication at the cell level. The study of how PrP polymorphisms influence the genetic control of prion propagation in cultured Rov cells may help elucidate basic mechanisms of prion replication.
- Published
- 2003
- Full Text
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21. Generation of a replication-competent, propagation-deficient virus vector based on the transmissible gastroenteritis coronavirus genome.
- Author
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Ortego J, Escors D, Laude H, and Enjuanes L
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- Animals, Cell Line, Cricetinae, Replicon, Sindbis Virus genetics, Transmissible gastroenteritis virus genetics, Transmissible gastroenteritis virus pathogenicity, Viral Envelope Proteins genetics, Genetic Vectors, Transmissible gastroenteritis virus metabolism, Viral Envelope Proteins metabolism, Virus Assembly, Virus Replication
- Abstract
Replication-competent propagation-deficient virus vectors based on the transmissible gastroenteritis coronavirus (TGEV) genome that are deficient in the essential E gene have been developed by complementation within E(+) packaging cell lines. Cell lines expressing the TGEV E protein were established using the noncytopathic Sindbis virus replicon pSINrep21. In addition, cell lines stably expressing the E gene under the CMV promoter have been developed. The Sindbis replicon vector and the ectopic TGEV E protein did not interfere with the rescue of infectious TGEV from full-length cDNA. Recombinant TGEV deficient in the nonessential 3a and 3b genes and the essential E gene (rTGEV-Delta3abDeltaE) was successfully rescued in these cell lines. rTGEV-Delta3abDeltaE reached high titers (10(7) PFU/ml) in baby hamster kidney cells expressing porcine aminopeptidase N (BHK-pAPN), the cellular receptor for TGEV, using Sindbis replicon and reached titers up to 5 x 10(5) PFU/ml in cells stably expressing E protein under the control of the CMV promoter. The virus titers were proportional to the E protein expression level. The rTGEV-Delta3abDeltaE virions produced in the packaging cell line showed the same morphology and stability under different pHs and temperatures as virus derived from the full-length rTGEV genome, although a delay in virus assembly was observed by electron microscopy and virus titration in the complementation system in relation to the wild-type virus. These viruses were stably grown for >10 passages in the E(+) packaging cell lines. The availability of packaging cell lines will significantly facilitate the production of safe TGEV-derived vectors for vaccination and possibly gene therapy.
- Published
- 2002
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22. Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins.
- Author
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Schwegmann-Wessels C, Zimmer G, Laude H, Enjuanes L, and Herrler G
- Subjects
- Animals, CD13 Antigens metabolism, Cell Line, Cell Membrane chemistry, Cell Membrane virology, Membrane Proteins chemistry, Membrane Proteins metabolism, Receptors, Virus metabolism, Sialoglycoproteins chemistry, Swine, Transmissible gastroenteritis virus genetics, Transmissible gastroenteritis virus pathogenicity, Sialoglycoproteins metabolism, Transmissible gastroenteritis virus metabolism
- Abstract
The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.
- Published
- 2002
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23. Organization of two transmissible gastroenteritis coronavirus membrane protein topologies within the virion and core.
- Author
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Escors D, Camafeita E, Ortego J, Laude H, and Enjuanes L
- Subjects
- Amino Acid Sequence, Animals, Glycoside Hydrolases pharmacology, Male, Molecular Sequence Data, Swine, Transmissible gastroenteritis virus chemistry, Viral Matrix Proteins chemistry, Virion chemistry
- Abstract
The difference in membrane (M) protein compositions between the transmissible gastroenteritis coronavirus (TGEV) virion and the core has been studied. The TGEV M protein adopts two topologies in the virus envelope, a Nexo-Cendo topology (with the amino terminus exposed to the virus surface and the carboxy terminus inside the virus particle) and a Nexo-Cexo topology (with both the amino and carboxy termini exposed to the virion surface). The existence of a population of M molecules adopting a Nexo-Cexo topology in the virion envelope was demonstrated by (i) immunopurification of (35)S-labeled TGEV virions using monoclonal antibodies (MAbs) specific for the M protein carboxy terminus (this immunopurification was inhibited only by deletion mutant M proteins that maintained an intact carboxy terminus), (ii) direct binding of M-specific MAbs to the virus surface, and (iii) mass spectrometry analysis of peptides released from trypsin-treated virions. Two-thirds of the total number of M protein molecules found in the virion were associated with the cores, and one-third was lost during core purification. MAbs specific for the M protein carboxy terminus were bound to native virions through the M protein in a Nexo-Cexo conformation, and these molecules were removed when the virus envelope was disrupted with NP-40 during virus core purification. All of the M protein was susceptible to N-glycosidase F treatment of the native virions, which indicates that all the M protein molecules are exposed to the virus surface. Cores purified from glycosidase-treated virions included M protein molecules that completely or partially lost the carbohydrate moiety, which strongly suggests that the M protein found in the cores was also exposed in the virus envelope and was not present exclusively in the virus interior. A TGEV virion structure integrating all the data is proposed. According to this working model, the TGEV virion consists of an internal core, made of the nucleocapsid and the carboxy terminus of the M protein, and the envelope, containing the spike (S) protein, the envelope (E) protein, and the M protein in two conformations. The two-thirds of the molecules that are in a Nexo-Cendo conformation (with their carboxy termini embedded within the virus core) interact with the internal core, and the remaining third of the molecules, whose carboxy termini are in a Nexo-Cexo conformation, are lost during virus core purification.
- Published
- 2001
- Full Text
- View/download PDF
24. Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp.
- Author
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Vilotte JL, Soulier S, Essalmani R, Stinnakre MG, Vaiman D, Lepourry L, Da Silva JC, Besnard N, Dawson M, Buschmann A, Groschup M, Petit S, Madelaine MF, Rakatobe S, Le Dur A, Vilette D, and Laude H
- Subjects
- Animals, Disease Susceptibility, Mice, Mice, Transgenic, PrPSc Proteins genetics, Scrapie pathology, Sheep, Transgenes, Disease Models, Animal, PrPSc Proteins toxicity, Scrapie etiology
- Abstract
The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.
- Published
- 2001
- Full Text
- View/download PDF
25. The membrane M protein carboxy terminus binds to transmissible gastroenteritis coronavirus core and contributes to core stability.
- Author
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Escors D, Ortego J, Laude H, and Enjuanes L
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal immunology, Epitope Mapping, Molecular Sequence Data, Nucleocapsid metabolism, Swine, Viral Matrix Proteins chemistry, Viral Matrix Proteins immunology, Transmissible gastroenteritis virus physiology, Viral Matrix Proteins physiology, Virus Assembly
- Abstract
The architecture of transmissible gastroenteritis coronavirus includes three different structural levels, the envelope, an internal core, and the nucleocapsid that is released when the core is disrupted. Starting from purified virions, core structures have been reproducibly isolated as independent entities. The cores were stabilized at basic pH and by the presence of divalent cations, with Mg(2+) ions more effectively contributing to core stability. Core structures showed high resistance to different concentrations of detergents, reducing agents, and urea and low concentrations of monovalent ions (<200 mM). Cores were composed of the nucleoprotein, RNA, and the C domain of the membrane (M) protein. At high salt concentrations (200 to 300 mM), the M protein was no longer associated with the nucleocapsid, which resulted in destruction of the core structure. A specific ionic interaction between the M protein carboxy terminus and the nucleocapsid was demonstrated using three complementary approaches: (i) a binding assay performed between a collection of M protein amino acid substitution or deletion mutants and purified nucleocapsids that led to the identification of a 16-amino-acid (aa) domain (aa 237 to 252) as being responsible for binding the M protein to the nucleocapsid; (ii) the specific inhibition of this binding by monoclonal antibodies (MAbs) binding to a carboxy-terminal M protein domain close to the indicated peptide but not by MAbs specific for the M protein amino terminus; and (iii) a 26-residue peptide, including the predicted sequence (aa 237 to 252), which specifically inhibited the binding. Direct binding of the M protein to the nucleoprotein was predicted, since degradation of the exposed RNA by RNase treatment did not affect the binding. It is proposed that the M protein is embedded within the virus membrane and that the C region, exposed to the interior face of the virion in a population of these molecules, interacts with the nucleocapsid to which it is anchored, forming the core. Only the C region of the M protein is part of the core.
- Published
- 2001
- Full Text
- View/download PDF
26. Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein.
- Author
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Nishida N, Harris DA, Vilette D, Laude H, Frobert Y, Grassi J, Casanova D, Milhavet O, and Lehmann S
- Subjects
- Animals, Mice, PrPSc Proteins genetics, Species Specificity, Tumor Cells, Cultured, Neuroblastoma virology, PrPSc Proteins pathogenicity, Prions genetics, Scrapie transmission
- Abstract
Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.
- Published
- 2000
- Full Text
- View/download PDF
27. Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes.
- Author
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Baudoux P, Carrat C, Besnardeau L, Charley B, and Laude H
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cattle, Coronavirus, Bovine genetics, Coronavirus, Bovine immunology, DNA Primers genetics, Humans, In Vitro Techniques, Leukocytes immunology, Microscopy, Electron, Molecular Sequence Data, Mutation, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Swine, Transmissible gastroenteritis virus immunology, Viral Matrix Proteins immunology, Viral Proteins immunology, Interferon-alpha biosynthesis, Transmissible gastroenteritis virus genetics, Transmissible gastroenteritis virus pathogenicity, Viral Matrix Proteins genetics, Viral Proteins genetics
- Abstract
Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-alpha synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-alpha-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-alpha almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-alpha is more likely to involve a specific, multimeric structure than a definite sequence motif.
- Published
- 1998
- Full Text
- View/download PDF
28. Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway.
- Author
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Eleouet JF, Chilmonczyk S, Besnardeau L, and Laude H
- Subjects
- Animals, Cell Line, Signal Transduction, Swine, Amino Acid Chloromethyl Ketones pharmacology, Apoptosis drug effects, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors pharmacology, Gastroenteritis, Transmissible, of Swine metabolism, Gastroenteritis, Transmissible, of Swine pathology, Transmissible gastroenteritis virus
- Abstract
In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-kappaB, a transcription factor known to be activated by oxidative stress, was examined. NF-kappaB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-kappaB activation is not critical for TGEV-induced apoptosis.
- Published
- 1998
- Full Text
- View/download PDF
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