{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T07:03:05Z","timestamp":1790838185034,"version":"4.1.0"},"update-to":[{"DOI":"10.1371\/journal.ppat.1011022","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,12,20]],"date-time":"2022-12-20T00:00:00Z","timestamp":1671494400000}}],"reference-count":47,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2022,12,8]],"date-time":"2022-12-08T00:00:00Z","timestamp":1670457600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100007492","name":"Fondation Bettencourt Schueller","doi-asserted-by":"publisher","award":["Prix Coup d'\u00e9lan pour la recherche fran\u00e7aise 2019"],"award-info":[{"award-number":["Prix Coup d'\u00e9lan pour la recherche fran\u00e7aise 2019"]}],"id":[{"id":"10.13039\/501100007492","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche, France","doi-asserted-by":"crossref","award":["ANR CE11, LiquidFact"],"award-info":[{"award-number":["ANR CE11, LiquidFact"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"crossref"}]},{"name":"FRM"},{"name":"France Bioimaging","award":["ANR\u201010\u2010INBS\u201004\u201001"],"award-info":[{"award-number":["ANR\u201010\u2010INBS\u201004\u201001"]}]}],"content-domain":{"domain":["www.plospathogens.org"],"crossmark-restriction":false},"short-container-title":["PLoS Pathog"],"abstract":"<jats:p>\n                    Rabies virus (RABV) transcription and replication take place within viral factories having liquid properties, called Negri bodies (NBs), that are formed by liquid-liquid phase separation (LLPS). The co-expression of RABV nucleoprotein (N) and phosphoprotein (P) in mammalian cells is sufficient to induce the formation of cytoplasmic biocondensates having properties that are like those of NBs. This cellular minimal system was previously used to identify P domains that are essential for biocondensates formation. Here, we constructed fluorescent versions of N and analyzed by FRAP their dynamics inside the biocondensates formed in this minimal system as well as in NBs of RABV-infected cells using FRAP. The behavior of N appears to be different of P as there was no fluorescence recovery of N proteins after photobleaching. We also identified arginine residues as well as two exposed loops of N involved in condensates formation. Corresponding N mutants exhibited distinct phenotypes in infected cells ranging from co-localization with NBs to exclusion from them associated with a dominant-negative effect on infection. We also demonstrated that\n                    <jats:italic>in vitro<\/jats:italic>\n                    , in crowded environments, purified P as well as purified N0-P complex (in which N is RNA-free) form liquid condensates. We identified P domains required for LLPS in this acellular system. P condensates were shown to associate with liposomes, concentrate RNA, and undergo a liquid-gel transition upon ageing. Conversely, N0-P droplets were disrupted upon incubation with RNA. Taken together, our data emphasize the central role of P in NBs formation and reveal some physicochemical features of P and N0-P droplets relevant for explaining NBs properties such as their envelopment by cellular membranes at late stages of infection and nucleocapsids ejections from the viral factories.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1011022","type":"journal-article","created":{"date-parts":[[2022,12,8]],"date-time":"2022-12-08T13:39:07Z","timestamp":1670506747000},"page":"e1011022","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":36,"title":["Properties of rabies virus phosphoprotein and nucleoprotein biocondensates formed in vitro and in cellulo"],"prefix":"10.1371","volume":"18","author":[{"given":"Quentin","family":"Nevers","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathalie","family":"Scrima","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Damien","family":"Glon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Romain","family":"Le Bars","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alice","family":"Decombe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nathalie","family":"Garnier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Malika","family":"Ouldali","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"C\u00e9cile","family":"Lagaudri\u00e8re-Gesbert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Danielle","family":"Blondel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aur\u00e9lie","family":"Albertini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0122-2954","authenticated-orcid":true,"given":"Yves","family":"Gaudin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,12,8]]},"reference":[{"issue":"4","key":"ppat.1011022.ref001","doi-asserted-by":"crossref","first-page":"e0003709","DOI":"10.1371\/journal.pntd.0003709","article-title":"Estimating the global burden of endemic canine rabies.","volume":"9","author":"K Hampson","year":"2015","journal-title":"PLoS Negl Trop Dis"},{"issue":"5785","key":"ppat.1011022.ref002","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1126\/science.1125280","article-title":"Crystal structure of the rabies virus nucleoprotein-RNA complex","volume":"313","author":"AA Albertini","year":"2006","journal-title":"Science"},{"key":"ppat.1011022.ref003","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/B978-0-12-387040-7.00001-9","article-title":"Rabies virus transcription and replication","volume":"79","author":"AA Albertini","year":"2011","journal-title":"Adv Virus Res"},{"issue":"20","key":"ppat.1011022.ref004","doi-asserted-by":"crossref","first-page":"10808","DOI":"10.1128\/JVI.00977-09","article-title":"Peptides that mimic the amino-terminal end of the rabies virus phosphoprotein have antiviral activity","volume":"83","author":"G Castel","year":"2009","journal-title":"J Virol"},{"issue":"2","key":"ppat.1011022.ref005","doi-asserted-by":"crossref","first-page":"422","DOI":"10.1016\/j.virol.2006.01.030","article-title":"Rabies virus chaperone: identification of the phosphoprotein peptide that keeps nucleoprotein soluble and free from non-specific RNA","volume":"349","author":"M Mavrakis","year":"2006","journal-title":"Virology"},{"issue":"16","key":"ppat.1011022.ref006","doi-asserted-by":"crossref","first-page":"7948","DOI":"10.1128\/JVI.00554-09","article-title":"Functional characterization of Negri bodies (NBs) in rabies virus-infected cells: Evidence that NBs are sites of viral transcription and replication.","volume":"83","author":"X Lahaye","year":"2009","journal-title":"J Virol"},{"issue":"1","key":"ppat.1011022.ref007","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1038\/s41467-017-00102-9","article-title":"Negri bodies are viral factories with properties of liquid organelles.","volume":"8","author":"J Nikolic","year":"2017","journal-title":"Nat Commun"},{"issue":"3","key":"ppat.1011022.ref008","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1016\/j.cell.2018.12.035","article-title":"Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates","volume":"176","author":"S Alberti","year":"2019","journal-title":"Cell"},{"issue":"5","key":"ppat.1011022.ref009","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1038\/nrm.2017.7","article-title":"Biomolecular condensates: organizers of cellular biochemistry","volume":"18","author":"SF Banani","year":"2017","journal-title":"Nat Rev Mol Cell Biol"},{"issue":"5935","key":"ppat.1011022.ref010","doi-asserted-by":"crossref","first-page":"1729","DOI":"10.1126\/science.1172046","article-title":"Germline P granules are liquid droplets that localize by controlled dissolution\/condensation","volume":"324","author":"CP Brangwynne","year":"2009","journal-title":"Science"},{"issue":"15","key":"ppat.1011022.ref011","doi-asserted-by":"crossref","first-page":"1603","DOI":"10.15252\/embj.201593517","article-title":"Droplet organelles?","volume":"35","author":"EM Courchaine","year":"2016","journal-title":"EMBO J"},{"key":"ppat.1011022.ref012","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.sbi.2016.10.015","article-title":"Intrinsically disordered proteins in overcrowded milieu: Membrane-less organelles, phase separation, and intrinsic disorder","volume":"44","author":"VN Uversky","year":"2017","journal-title":"Curr Opin Struct Biol"},{"issue":"5","key":"ppat.1011022.ref013","doi-asserted-by":"crossref","DOI":"10.1128\/mBio.02290-17","article-title":"Phase Transitions Drive the Formation of Vesicular Stomatitis Virus Replication Compartments.","volume":"9","author":"BS 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Galloux","year":"2020","journal-title":"mBio"},{"issue":"12","key":"ppat.1011022.ref017","doi-asserted-by":"crossref","first-page":"118831","DOI":"10.1016\/j.bbamcr.2020.118831","article-title":"Negri bodies and other virus membrane-less replication compartments","volume":"1867","author":"Q Nevers","year":"2020","journal-title":"Biochim Biophys Acta Mol Cell Res"},{"issue":"4","key":"ppat.1011022.ref018","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1128\/jvi.14.4.981-996.1974","article-title":"Further studies on the replication of rabies and rabies-like viruses in organized cultures of mammalian neural tissues","volume":"14","author":"S Matsumoto","year":"1974","journal-title":"J Virol"},{"issue":"5","key":"ppat.1011022.ref019","doi-asserted-by":"crossref","first-page":"978","DOI":"10.1016\/j.jmb.2009.03.061","article-title":"Modular organization of rabies virus phosphoprotein","volume":"388","author":"FC Gerard","year":"2009","journal-title":"J Mol 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Nott","year":"2015","journal-title":"Molecular cell"},{"issue":"2","key":"ppat.1011022.ref029","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1016\/j.cell.2020.03.046","article-title":"G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules","volume":"181","author":"P Yang","year":"2020","journal-title":"Cell"},{"issue":"3","key":"ppat.1011022.ref030","doi-asserted-by":"crossref","first-page":"e0109922","DOI":"10.1128\/mbio.01099-22","article-title":"Human Metapneumovirus Phosphoprotein Independently Drives Phase Separation and Recruits Nucleoprotein to Liquid-Like Bodies.","volume":"13","author":"KB Boggs","year":"2022","journal-title":"mBio"},{"issue":"1","key":"ppat.1011022.ref031","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1016\/j.cell.2015.09.015","article-title":"Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization","volume":"163","author":"A Molliex","year":"2015","journal-title":"Cell"},{"issue":"1","key":"ppat.1011022.ref032","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1038\/s41467-017-00655-9","article-title":"Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus.","volume":"8","author":"V Rincheval","year":"2017","journal-title":"Nat Commun"},{"key":"ppat.1011022.ref033","article-title":"Host casein kinase 1-mediated phosphorylation modulates phase separation of a rhabdovirus phosphoprotein and virus infection.","volume":"11","author":"XD Fang","year":"2022","journal-title":"Elife"},{"key":"ppat.1011022.ref034","doi-asserted-by":"crossref","DOI":"10.7554\/eLife.30294","article-title":"Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins","volume":"6","author":"TS Harmon","year":"2017","journal-title":"Elife"},{"issue":"6403","key":"ppat.1011022.ref035","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1126\/science.aat1022","article-title":"DNA-induced liquid phase condensation of cGAS activates innate immune signaling","volume":"361","author":"M Du","year":"2018","journal-title":"Science"},{"issue":"2","key":"ppat.1011022.ref036","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1016\/j.molcel.2015.08.018","article-title":"Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins","volume":"60","author":"Y Lin","year":"2015","journal-title":"Molecular cell"},{"issue":"12","key":"ppat.1011022.ref037","doi-asserted-by":"crossref","first-page":"E2466","DOI":"10.1073\/pnas.1614462114","article-title":"Amyotrophic lateral sclerosis-linked mutations increase the viscosity of liquid-like TDP-43 RNP granules in neurons","volume":"114","author":"PP Gopal","year":"2017","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"2","key":"ppat.1011022.ref038","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1016\/j.neuron.2019.02.038","article-title":"Cytoplasmic TDP-43 De-mixing Independent of Stress Granules Drives Inhibition of Nuclear Import, Loss of Nuclear TDP-43, and Cell Death","volume":"102","author":"F Gasset-Rosa","year":"2019","journal-title":"Neuron"},{"issue":"10","key":"ppat.1011022.ref039","doi-asserted-by":"crossref","DOI":"10.1083\/jcb.202103175","article-title":"Intracellular wetting mediates contacts between liquid compartments and membrane-bound organelles","volume":"220","author":"H Kusumaatmaja","year":"2021","journal-title":"J Cell Biol"},{"issue":"6402","key":"ppat.1011022.ref040","doi-asserted-by":"crossref","first-page":"604","DOI":"10.1126\/science.aat5671","article-title":"A liquid phase of synapsin and lipid vesicles","volume":"361","author":"D Milovanovic","year":"2018","journal-title":"Science"},{"issue":"1","key":"ppat.1011022.ref041","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1038\/s41467-020-20462-z","article-title":"Cooperative function of synaptophysin and synapsin in the generation of synaptic vesicle-like clusters in non-neuronal cells.","volume":"12","author":"D Park","year":"2021","journal-title":"Nat Commun"},{"issue":"8","key":"ppat.1011022.ref042","doi-asserted-by":"crossref","first-page":"2594","DOI":"10.1016\/j.celrep.2020.01.092","article-title":"Vesicle Clustering in a Living Synapse Depends on a Synapsin Region that Mediates Phase Separation.","volume":"30","author":"A Pechstein","year":"2020","journal-title":"Cell Rep"},{"issue":"7868","key":"ppat.1011022.ref043","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1038\/s41586-021-03703-z","article-title":"A condensate-hardening drug blocks RSV replication in vivo","volume":"595","author":"J Risso-Ballester","year":"2021","journal-title":"Nature"},{"issue":"1","key":"ppat.1011022.ref044","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1128\/JVI.73.1.251-259.1999","article-title":"Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.","volume":"73","author":"UJ Buchholz","year":"1999","journal-title":"J Virol"},{"issue":"5","key":"ppat.1011022.ref045","doi-asserted-by":"crossref","first-page":"3818","DOI":"10.1128\/JVI.73.5.3818-3825.1999","article-title":"Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3\u2019 copy-back ambisense rabies virus","volume":"73","author":"S Finke","year":"1999","journal-title":"J Virol"},{"issue":"(Pt 1","key":"ppat.1011022.ref046","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1099\/0022-1317-78-1-119","article-title":"Mapping of monoclonal antibody epitopes of the rabies virus P protein","volume":"78","author":"H Raux","year":"1997","journal-title":"J Gen Virol"},{"issue":"7","key":"ppat.1011022.ref047","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis.","volume":"9","author":"J Schindelin","year":"2012","journal-title":"Nat Methods"}],"updated-by":[{"DOI":"10.1371\/journal.ppat.1011022","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,12,20]],"date-time":"2022-12-20T00:00:00Z","timestamp":1671494400000}}],"container-title":["PLOS 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