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Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate Nucleocapsid transport
Proceedings of the National Academy of Sciences, 2018Co-Authors: Yuki Takamatsu, Larissa Kolesnikova, Stephan BeckerAbstract:The intracytoplasmic movement of Nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral Nucleocapsid transport competency is complicated by the dynamic and complex nature of Nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV Nucleocapsid-like structures with different protein compositions. Only three of the five EBOV Nucleocapsid proteins—nucleoprotein, VP35, and VP24—were necessary and sufficient to form transport-competent Nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of Nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of Nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of Nucleocapsid recruitment into filopodia, the sites of EBOV budding.
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Structure and assembly of the Ebola virus Nucleocapsid
Nature, 2017Co-Authors: Larissa Kolesnikova, Stephan Becker, Takeshi Noda, Mairi Clarke, Alexander Koehler, John A. G. BriggsAbstract:The Ebola virus Nucleocapsid—a protein shell—encloses, or 'encapsidates', the viral genome and acts as a scaffold for virus assembly and as a template for genome replication. John Briggs and colleagues use cryo-electron tomography to solve the structure of the Nucleocapsid of the Ebola virus. They use the structures of the Ebola virus Nucleocapsid within intact viruses and recombinant assemblies to propose a model for viral RNA encapsidation and accessory protein recruitment. Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever^ 1 . Filoviruses are within the order Mononegavirales^ 2 , which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical Nucleocapsid. The Nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein–nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein^ 3 , 4 , 5 , 6 , and cryo-electron microscopy of Nucleocapsid^ 7 , 8 , 9 , 10 , 11 , 12 or Nucleocapsid-like structures^ 11 , 12 , 13 . There have been no high-resolution reconstructions of complete mononegavirus Nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus Nucleocapsid within intact viruses and recombinant Nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the Nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, Nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment. Application of cryo-electron tomography and subtomogram averaging to determine the structure of the Ebola virus Nucleocapsid within intact viruses and recombinant Nucleocapsid-like assemblies.
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Structure and assembly of the Ebola virus Nucleocapsid
Nature, 2017Co-Authors: William Wan, Larissa Kolesnikova, Stephan Becker, Takeshi Noda, Mairi Clarke, Alexander Koehler, John A. G. BriggsAbstract:Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical Nucleocapsid. The Nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of Nucleocapsid or Nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus Nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus Nucleocapsid within intact viruses and recombinant Nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the Nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, Nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.
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live cell imaging of marburg virus infected cells uncovers actin dependent transport of Nucleocapsids over long distances
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Gordian Schudt, Olga Dolnik, Beate Sodeik, Larissa Kolesnikova, Stephan BeckerAbstract:Transport of large viral Nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of Nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of Nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of Nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the Nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, Nucleocapsids were transported at ∼200 nm/s; (ii) Nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated Nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of Nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.
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phosphorylation of marburg virus matrix protein vp40 triggers assembly of Nucleocapsids with the viral envelope at the plasma membrane
Cellular Microbiology, 2012Co-Authors: Larissa Kolesnikova, Gordian Schudt, Evamaria Mittler, Hosam Shamseldin, Stephan BeckerAbstract:Summary Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting Nucleocapsids and the surface protein GP to filopodia, the sites of viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus-like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non-phosphorylatable form of VP40 (VP40mut). VP40mut was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon-induced gene expression similarly to wild-type VP40. However, VP40mut was specifically impaired in its ability to recruit Nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of Nucleocapsids to the viral envelope.
Larissa Kolesnikova - One of the best experts on this subject based on the ideXlab platform.
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Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate Nucleocapsid transport
Proceedings of the National Academy of Sciences, 2018Co-Authors: Yuki Takamatsu, Larissa Kolesnikova, Stephan BeckerAbstract:The intracytoplasmic movement of Nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral Nucleocapsid transport competency is complicated by the dynamic and complex nature of Nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV Nucleocapsid-like structures with different protein compositions. Only three of the five EBOV Nucleocapsid proteins—nucleoprotein, VP35, and VP24—were necessary and sufficient to form transport-competent Nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of Nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of Nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of Nucleocapsid recruitment into filopodia, the sites of EBOV budding.
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Structure and assembly of the Ebola virus Nucleocapsid
Nature, 2017Co-Authors: Larissa Kolesnikova, Stephan Becker, Takeshi Noda, Mairi Clarke, Alexander Koehler, John A. G. BriggsAbstract:The Ebola virus Nucleocapsid—a protein shell—encloses, or 'encapsidates', the viral genome and acts as a scaffold for virus assembly and as a template for genome replication. John Briggs and colleagues use cryo-electron tomography to solve the structure of the Nucleocapsid of the Ebola virus. They use the structures of the Ebola virus Nucleocapsid within intact viruses and recombinant assemblies to propose a model for viral RNA encapsidation and accessory protein recruitment. Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever^ 1 . Filoviruses are within the order Mononegavirales^ 2 , which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical Nucleocapsid. The Nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein–nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein^ 3 , 4 , 5 , 6 , and cryo-electron microscopy of Nucleocapsid^ 7 , 8 , 9 , 10 , 11 , 12 or Nucleocapsid-like structures^ 11 , 12 , 13 . There have been no high-resolution reconstructions of complete mononegavirus Nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus Nucleocapsid within intact viruses and recombinant Nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the Nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, Nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment. Application of cryo-electron tomography and subtomogram averaging to determine the structure of the Ebola virus Nucleocapsid within intact viruses and recombinant Nucleocapsid-like assemblies.
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Structure and assembly of the Ebola virus Nucleocapsid
Nature, 2017Co-Authors: William Wan, Larissa Kolesnikova, Stephan Becker, Takeshi Noda, Mairi Clarke, Alexander Koehler, John A. G. BriggsAbstract:Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause haemorrhagic fever. Filoviruses are within the order Mononegavirales, which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein and other viral proteins to form a helical Nucleocapsid. The Nucleocapsid acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into nucleoprotein-nucleoprotein interactions have been derived from structural studies of oligomerized, RNA-encapsidating nucleoprotein, and cryo-electron microscopy of Nucleocapsid or Nucleocapsid-like structures. There have been no high-resolution reconstructions of complete mononegavirus Nucleocapsids. Here we apply cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus Nucleocapsid within intact viruses and recombinant Nucleocapsid-like assemblies. These structures reveal the identity and arrangement of the Nucleocapsid components, and suggest that the formation of an extended α-helix from the disordered carboxy-terminal region of nucleoprotein-core links nucleoprotein oligomerization, Nucleocapsid condensation, RNA encapsidation, and accessory protein recruitment.
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live cell imaging of marburg virus infected cells uncovers actin dependent transport of Nucleocapsids over long distances
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Gordian Schudt, Olga Dolnik, Beate Sodeik, Larissa Kolesnikova, Stephan BeckerAbstract:Transport of large viral Nucleocapsids from replication centers to assembly sites requires contributions from the host cytoskeleton via cellular adaptor and motor proteins. For the Marburg and Ebola viruses, related viruses that cause severe hemorrhagic fevers, the mechanism of Nucleocapsid transport remains poorly understood. Here we developed and used live-cell imaging of fluorescently labeled viral and host proteins to characterize the dynamics and molecular requirements of Nucleocapsid transport in Marburg virus-infected cells under biosafety level 4 conditions. The study showed a complex actin-based transport of Nucleocapsids over long distances from the viral replication centers to the budding sites. Only after the Nucleocapsids had associated with the matrix viral protein VP40 at the plasma membrane were they recruited into filopodia and cotransported with host motor myosin 10 toward the budding sites at the tip or side of the long cellular protrusions. Three different transport modes and velocities were identified: (i) Along actin filaments in the cytosol, Nucleocapsids were transported at ∼200 nm/s; (ii) Nucleocapsids migrated from one actin filament to another at ∼400 nm/s; and (iii) VP40-associated Nucleocapsids moved inside filopodia at 100 nm/s. Unique insights into the spatiotemporal dynamics of Nucleocapsids and their interaction with the cytoskeleton and motor proteins can lead to novel classes of antivirals that interfere with the trafficking and subsequent release of the Marburg virus from infected cells.
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phosphorylation of marburg virus matrix protein vp40 triggers assembly of Nucleocapsids with the viral envelope at the plasma membrane
Cellular Microbiology, 2012Co-Authors: Larissa Kolesnikova, Gordian Schudt, Evamaria Mittler, Hosam Shamseldin, Stephan BeckerAbstract:Summary Marburg virus (MARV) matrix protein VP40 plays a key role in virus assembly, recruiting Nucleocapsids and the surface protein GP to filopodia, the sites of viral budding. In addition, VP40 is the only MARV protein able to induce the release of filamentous virus-like particles (VLPs) indicating its function in MARV budding. Here, we demonstrated that VP40 is phosphorylated and that tyrosine residues at positions 7, 10, 13 and 19 represent major phosphorylation acceptor sites. Mutagenesis of these tyrosine residues resulted in expression of a non-phosphorylatable form of VP40 (VP40mut). VP40mut was able to bind to cellular membranes, produce filamentous VLPs, and inhibit interferon-induced gene expression similarly to wild-type VP40. However, VP40mut was specifically impaired in its ability to recruit Nucleocapsid structures into filopodia, and released infectious VLPs (iVLPs) had low infectivity. These results indicated that tyrosine phosphorylation of VP40 is important for triggering the recruitment of Nucleocapsids to the viral envelope.
Rob W H Ruigrok - One of the best experts on this subject based on the ideXlab platform.
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Assembly and cryo-EM structures of RNA-specific measles virus Nucleocapsids provide mechanistic insight into paramyxoviral replication
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Ambroise Desfosses, Guy Schoehn, Rob W H Ruigrok, Irina Gutsche, Malene Ringkjøbing Jensen, Sigrid Milles, Serafima Guseva, Jacques-philippe Colletier, Damien Maurin, Martin BlackledgeAbstract:Assembly of paramyxoviral Nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus Nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within Nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles Nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for Nucleocapsid-like particle assembly.
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Structural virology. Near-atomic cryo-EM structure of the helical measles virus Nucleocapsid.
Science, 2015Co-Authors: Irina Gutsche, Rob W H Ruigrok, Ambroise Desfosses, Grégory Effantin, Wai-li Ling, Melina Haupt, Carsten Sachse, Guy SchoehnAbstract:Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical Nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus Nucleocapsid offers important insights into the mechanism of the helical polymerization of Nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design.
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near atomic cryo em structure of the helical measles virus Nucleocapsid
Science, 2015Co-Authors: Irina Gutsche, Rob W H Ruigrok, Ambroise Desfosses, Grégory Effantin, Wai-li Ling, Melina Haupt, Carsten Sachse, Guy SchoehnAbstract:Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical Nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus Nucleocapsid offers important insights into the mechanism of the helical polymerization of Nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design.
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Atomic resolution description of the interaction between the nucleoprotein and phosphoprotein of Hendra virus.
PLoS Pathogens, 2013Co-Authors: Guillaume Communie, Rob W H Ruigrok, Johnny Habchi, Filip Yabukarski, David Blocquel, Robert Schneider, Nicolas Tarbouriech, Nicolas Papageorgiou, Marc Jamin, Malene Ringkjøbing JensenAbstract:Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical Nucleocapsid. Recruitment of the viral polymerase onto the Nucleocapsid template relies on the interaction between the C-terminal domain, N(TAIL), of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered N(TAIL) domain in its isolated state and in intact Nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV Nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between N(TAIL) and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of N(TAIL) upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact Nucleocapsids and XD. Our results indicate that monomeric XD binds to N(TAIL) without triggering an additional unwinding of the Nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact Nucleocapsids in Paramyxoviridae.
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Self-organization of the vesicular stomatitis virus Nucleocapsid into a bullet shape.
Nature Communications, 2013Co-Authors: Ambroise Desfosses, Guy Schoehn, Rob W H Ruigrok, Danielle Blondel, Marc Jamin, Euripedes A Ribeiro, Delphine Guilligay, Irina GutscheAbstract:The typical bullet shape of Rhabdoviruses is thought to rely on the matrix protein for stabilizing the Nucleocapsid coil. Here we scrutinize the morphology of purified and recombinant Nucleocapsids of vesicular stomatitis virus in vitro. We elucidate pH and ionic strength conditions for their folding into conical tips and further growth into whole bullets, and provide cryo-electron microscopy reconstructions of the bullet tip and the helical trunk. We address conformational variability of the reconstituted Nucleocapsids and the issue of constraints imposed by the binding of matrix protein. Our findings bridge the gap between the isolated nucleoprotein-RNA string in its form of an undulating ribbon, and the tight bullet-shaped virion skeleton.
Brett D Lindenbach - One of the best experts on this subject based on the ideXlab platform.
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a sensitive yellow fever virus entry reporter identifies valosin containing protein vcp p97 as an essential host factor for flavivirus uncoating
Mbio, 2020Co-Authors: Harish N. Ramanathan, Florian Douam, Priscilla L Yang, John W Schoggins, Alexander Ploss, Jinhong Chang, Shuo Zhang, Brett D LindenbachAbstract:ABSTRACT While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as Nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for Nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus Nucleocapsids during a postfusion step in virus entry. IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus Nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that Nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
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a sensitive yellow fever virus entry reporter identifies valosin containing protein vcp p97 as an essential host factor for flavivirus uncoating
bioRxiv, 2019Co-Authors: Harish N. Ramanathan, Florian Douam, Priscilla L Yang, Alexander Ploss, Jinhong Chang, Shuo Zhang, Brett D LindenbachAbstract:While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the post-fusion events that precede RNA replication, such as Nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFV{Delta}SK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFV{Delta}SK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E expression; however, as expected, gene expression from the defective reporter virus was insensitive to a small molecule inhibitor of YFV RNA replication. YFV{Delta}SK/Nluc gene expression was also shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for Nucleocapsid uncoating to occur, as well as valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large macromolecular complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a post-fusion, pre-translation step in YFV entry. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus Nucleocapsids during a post-fusion step in virus entry.nnIMPORTANCEFlaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus Nucleocapsids are disassembled during virus entry remain unclear. Here we show that the yellow fever virus Nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.
Irina Gutsche - One of the best experts on this subject based on the ideXlab platform.
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Assembly and cryo-EM structures of RNA-specific measles virus Nucleocapsids provide mechanistic insight into paramyxoviral replication
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Ambroise Desfosses, Guy Schoehn, Rob W H Ruigrok, Irina Gutsche, Malene Ringkjøbing Jensen, Sigrid Milles, Serafima Guseva, Jacques-philippe Colletier, Damien Maurin, Martin BlackledgeAbstract:Assembly of paramyxoviral Nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus Nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within Nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles Nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for Nucleocapsid-like particle assembly.
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High resolution cryo-EM structure of the helical RNA-bound Hantaan virus Nucleocapsid reveals its assembly mechanisms
eLife, 2019Co-Authors: Benoît Arragain, Guy Schoehn, Juan Reguera, Ambroise Desfosses, Irina Gutsche, Hélène MaletAbstract:Negative-strand RNA viruses condense their genome into helical Nucleocapsids that constitute essential templates for viral replication and transcription. The intrinsic flexibility of Nucleocapsids usually prevents their full-length structural characterization at high resolution. Here we describe purification of full-length recombinant metastable helical Nucleocapsid of Hantaan virus ($Hantaviridae$ family, $Bunyavirales$ order) and determine its structure at 3.3 Å resolution by cryo-electron microscopy. The structure reveals the mechanisms of helical multimerization via sub-domain exchanges between protomers and highlights nucleotide positions in a continuous positively charged groove compatible with viral genome binding. It uncovers key sites for future structure-based design of antivirals that are currently lacking to counteract life-threatening hantavirus infections. The structure also suggests a model of nucleoprotein-polymerase interaction that would enable replication and transcription solely upon local disruption of the Nucleocapsid.
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Structural virology. Near-atomic cryo-EM structure of the helical measles virus Nucleocapsid.
Science, 2015Co-Authors: Irina Gutsche, Rob W H Ruigrok, Ambroise Desfosses, Grégory Effantin, Wai-li Ling, Melina Haupt, Carsten Sachse, Guy SchoehnAbstract:Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical Nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus Nucleocapsid offers important insights into the mechanism of the helical polymerization of Nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design.
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near atomic cryo em structure of the helical measles virus Nucleocapsid
Science, 2015Co-Authors: Irina Gutsche, Rob W H Ruigrok, Ambroise Desfosses, Grégory Effantin, Wai-li Ling, Melina Haupt, Carsten Sachse, Guy SchoehnAbstract:Measles is a highly contagious human disease. We used cryo-electron microscopy and single particle-based helical image analysis to determine the structure of the helical Nucleocapsid formed by the folded domain of the measles virus nucleoprotein encapsidating an RNA at a resolution of 4.3 angstroms. The resulting pseudoatomic model of the measles virus Nucleocapsid offers important insights into the mechanism of the helical polymerization of Nucleocapsids of negative-strand RNA viruses, in particular via the exchange subdomains of the nucleoprotein. The structure reveals the mode of the nucleoprotein-RNA interaction and explains why each nucleoprotein of measles virus binds six nucleotides, whereas the respiratory syncytial virus nucleoprotein binds seven. It provides a rational basis for further analysis of measles virus replication and transcription, and reveals potential targets for drug design.
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Self-organization of the vesicular stomatitis virus Nucleocapsid into a bullet shape.
Nature Communications, 2013Co-Authors: Ambroise Desfosses, Guy Schoehn, Rob W H Ruigrok, Danielle Blondel, Marc Jamin, Euripedes A Ribeiro, Delphine Guilligay, Irina GutscheAbstract:The typical bullet shape of Rhabdoviruses is thought to rely on the matrix protein for stabilizing the Nucleocapsid coil. Here we scrutinize the morphology of purified and recombinant Nucleocapsids of vesicular stomatitis virus in vitro. We elucidate pH and ionic strength conditions for their folding into conical tips and further growth into whole bullets, and provide cryo-electron microscopy reconstructions of the bullet tip and the helical trunk. We address conformational variability of the reconstituted Nucleocapsids and the issue of constraints imposed by the binding of matrix protein. Our findings bridge the gap between the isolated nucleoprotein-RNA string in its form of an undulating ribbon, and the tight bullet-shaped virion skeleton.
