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Yoshihiro Kawaoka - One of the best experts on this subject based on the ideXlab platform.
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cryo em structure of the ebola Virus Nucleoprotein rna complex at 3 6 a resolution
Nature, 2018Co-Authors: Takeshi Noda, Yoshihiro Kawaoka, Yukihiko Sugita, Hideyuki Matsunami, Matthias WolfAbstract:Ebola Virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are Viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical Nucleoprotein–RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The Nucleoprotein–RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and Nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola Virus Nucleoprotein–RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola Virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by Nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of Nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development. Near-atomic resolution cryo-electron microscopy structures of the Zaire ebolaVirus Nucleoprotein indicate a complex transition from the RNA-free to RNA-bound forms of the protein, and reveal the mechanism of oligomer formation and helical assembly.
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characterization of the ebola Virus Nucleoprotein rna complex
Journal of General Virology, 2010Co-Authors: Takeshi Noda, Yoshihiro Kawaoka, Kyoji Hagiwara, Hiroshi SagaraAbstract:When Ebola Virus Nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Here, the recombinant NP helix purified from cells expressing NP was characterized biochemically and morphologically. We found that the recombinant NP helix is associated with non-viral RNA, which is not protected from RNase digestion and that the morphology of the helix changes depending on the environmental salt concentration. The N-terminal 450 aa residues of NP are sufficient for these properties. However, digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that this RNA is an essential structural component of the helix, binding to individual NP molecules via the N-terminal 450 aa. These findings enhance our knowledge of Ebola Virus assembly and understanding of the Ebola Virus life cycle.
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mutational analysis of conserved amino acids in the influenza a Virus Nucleoprotein
Journal of Virology, 2009Co-Authors: Tokiko Watanabe, Makoto Ozawa, Shinya Yamada, Masato Hatta, Shinji Watanabe, Asuka Nanbo, Satoshi Kakugawa, Masayuki Shimojima, Gabriele Neumann, Yoshihiro KawaokaAbstract:The Nucleoprotein (NP), which has multiple functions during the Virus life cycle, possesses regions that are highly conserved among influenza A, B, and C Viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 Viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant Viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of Viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.
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Contributions of Two Nuclear Localization Signals of Influenza A Virus Nucleoprotein to Viral Replication
Journal of virology, 2006Co-Authors: Makoto Ozawa, Ken Fujii, Yukiko Muramoto, Shinya Yamada, Seiya Yamayoshi, Ayato Takada, Hideo Goto, Taisuke Horimoto, Yoshihiro KawaokaAbstract:The RNA genome of influenza A Virus, which forms viral riboNucleoprotein complexes (vRNPs) with viral polymerase subunit proteins (PA, PB1, and PB2) and Nucleoprotein (NP), is transcribed and replicated in the nucleus. NP, the major component of vRNPs, has at least two amino acid sequences that serve as nuclear localization signals (NLSs): an unconventional NLS (residues 3 to 13; NLS1) and a bipartite NLS (residues 198 to 216; NLS2). Although both NLSs are known to play a role in nuclear transport, their relative contributions to viral replication are poorly understood. We therefore investigated their contributions to NP subcellular/subnuclear localization, viral RNA (vRNA) transcription, and viral replication. Abolishing the unconventional NLS caused NP to localize predominantly to the cytoplasm and affected its activity in vRNA transcription. However, we were able to create a Virus whose NP contained amino acid substitutions in NLS1 known to abolish its nuclear localization function, although this Virus was highly attenuated. These results indicate that while the unconventional NLS is not essential for viral replication, it is necessary for efficient viral mRNA synthesis. On the other hand, the bipartite NLS, whose contribution to the nuclear transport of NP is limited, was essential for vRNA transcription and NP's nucleolar accumulation. A Virus with nonfunctional NLS2 could not be generated. Thus, the bipartite NLS, but not the unconventional NLS, of NP is essential for influenza A Virus replication.
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nuclear import and export of influenza Virus Nucleoprotein
Journal of Virology, 1997Co-Authors: G Neumann, Maria R Castrucci, Yoshihiro KawaokaAbstract:Influenza Virus Nucleoprotein (NP) shuttles between the nucleus and the cytoplasm. A nuclear localization signal (NLS) has been identified in NP at amino acids 327 to 345 (J. Davey et al., Cell 40:667-675, 1985). However, some NP mutants that lack this region still localize to the nucleus, suggesting an additional NLS in NP. We therefore investigated the nucleocytoplasmic transport of NP from influenza Virus A/WSN/33 (H1N1). NP deletion constructs lacking the 38 N-terminal amino acids, as well as those lacking the 38 N-terminal amino acids and the previously identified NLS, localized to both the cytoplasm and the nucleus. Nuclear localization of a protein containing amino acids 1 to 38 of NP fused to LacZ proved that these 38 amino acids function as an NLS. Within this region, we identified two basic amino acids, Lys7 and Arg8, that are crucial for NP nuclear import. After being imported into the nucleus, the wild-type NP and the NP-LacZ fusion construct containing amino acids 1 to 38 of NP were both transported back to the cytoplasm, where they accumulated. These data indicate that NP has intrinsic structural features that allow nuclear import, nuclear export, and cytoplasmic accumulation in the absence of any other viral proteins. Further, the information required for nuclear import and export is located in the 38 N-terminal amino acids of NP, although other NP nuclear export signals may exist. Treatment of cells with a protein kinase C inhibitor increased the amounts of nuclear NP, whereas treatment of cells with a phosphorylation stimulator increased the amounts of cytoplasmic NP. These findings suggest a role of phosphorylation in nucleocytoplasmic transport of NP.
Guy Schoehn - One of the best experts on this subject based on the ideXlab platform.
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near atomic cryo em structure of the helical measles Virus nucleocapsid
Science, 2015Co-Authors: Irina Gutsche, 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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rna induced polymerization of the borna disease Virus Nucleoprotein
Virology, 2010Co-Authors: Miriam Hock, Guy Schoehn, Ina Kraus, Wolfgang Garten, Marc Jamin, Cornelia Andreiselmer, Winfried WeissenhornAbstract:The Borna disease Virus (BDV) Nucleoprotein (N) monomer resembles the Nucleoprotein structures from rabies Virus (RABV) and vesicular stomatitis Virus (VSV). We show that BDV N assembles into ring- and string-like structures in the presence of 5' genomic BDV RNA. RNA induced polymerization is partly RNA-specific since polymerization is inefficient in the presence of 3' genomic BDV RNA or E. coli RNA. Mutagenesis of basic residues located in the cleft made up by the N- and C-terminal domains of N abrogate RNA-induced polymerization indicating that BDV N binds RNA similarly as observed in case of RABV and VSV N-RNA complexes. Bound RNA is not protected and sensitive to degradation. N-RNA polymers form complexes with the phosphoprotein P as required for functional transcription or replication units. Our data indicate that BDV N utilizes similar structural principles for N-RNA and N-P-RNA complex formation as observed for related negative strand RNA Viruses.
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crystal structure of the rabies Virus Nucleoprotein rna complex
Science, 2006Co-Authors: Aurélie A V Albertini, Amy K Wernimont, Tadeusz Muziol, Raimond B G Ravelli, Cedric R Clapier, Guy SchoehnAbstract:Negative-strand RNA Viruses condense their genome into a helical Nucleoprotein-RNA complex, the nucleocapsid, which is packed into virions and serves as a template for the RNA-dependent RNA polymerase complex. The crystal structure of a recombinant rabies Virus Nucleoprotein-RNA complex, organized in an undecameric ring, has been determined at 3.5 angstrom resolution. Polymerization of the Nucleoprotein is achieved by domain exchange between protomers, with flexible hinges allowing nucleocapsid formation. The two core domains of the Nucleoprotein clamp around the RNA at their interface and shield it from the environment. RNA sequestering by Nucleoproteins is likely a common mechanism used by negative-strand RNA Viruses to protect their genomes from the innate immune response directed against viral RNA in human host cells at certain stages of an infectious cycle.
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structure of recombinant rabies Virus Nucleoprotein rna complex and identification of the phosphoprotein binding site
Journal of Virology, 2001Co-Authors: Guy Schoehn, Danielle Blondel, Frédéric Iseni, Manos Mavrakis, Rob W H RuigrokAbstract:Rabies Virus Nucleoprotein (N) was produced in insect cells, in which it forms Nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies Virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the Nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies Virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies Virus N-RNA and from nucleocapsid in Virus, we propose a low-resolution model for rabies Virus N-RNA in the Virus.
Rob W H Ruigrok - One of the best experts on this subject based on the ideXlab platform.
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Influenza Virus Nucleoprotein: structure, RNA binding, oligomerization and antiviral drug target
Future Microbiology, 2013Co-Authors: Sylvie Chenavas, Rob W H Ruigrok, Thibaut Crépin, Bernard Delmas, Anny Slama-schwokAbstract:The Nucleoprotein (NP) of influenza Virus covers the viral RNA entirely and it is this NP-RNA complex that is the template for transcription and replication by the viral polymerase. Purified NP forms a dynamic equilibrium between monomers and small oligomers, but only the monomers can oligomerize onto RNA. Therefore, drugs that stabilize the monomers or that induce abnormal oligomerization may have an antiviral effect, as would drugs that interfere with RNA binding. Crystal structures have been produced for monomeric and dimeric mutants, and for trimers and tetramers; high-resolution electron microscopy structures have also been calculated for the viral NP-RNA complex. We explain how these structures and the dynamic oligomerization equilibrium of NP can be and have been used for anti-influenza drug development.
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Nucleoprotein rna orientation in the measles Virus nucleocapsid by three dimensional electron microscopy
Journal of Virology, 2011Co-Authors: Ambroise Desfosses, Rob W H Ruigrok, Gael Goret, Leandro F Estrozi, Irina GutscheAbstract:Recombinant measles Virus Nucleoprotein-RNA (N-RNA) helices were analyzed by negative-stain electron microscopy. Three-dimensional reconstructions of trypsin-digested and intact nucleocapsids coupled to the docking of the atomic structure of the respiratory syncytial Virus (RSV) N-RNA subunit into the electron microscopy density map support a model that places the RNA at the exterior of the helix and the disordered C-terminal domain toward the helix interior, and they suggest the position of the six nucleotides with respect to the measles N protomer.
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structural disorder within sendai Virus Nucleoprotein and phosphoprotein insight into the structural basis of molecular recognition
Protein and Peptide Letters, 2010Co-Authors: Malene Ringkjobing Jensen, Rob W H Ruigrok, Klaartje Houben, Laurence Blanchard, Pau Bernado, Dominque Marion, Martin BlackledgeAbstract:Intrinsically disordered regions of significant length are present throughout eukaryotic genomes, and are particularly prevalent in viral proteins. Due to their inherent flexibility, these proteins inhabit a conformational landscape that is too complex to be described by classical structural biology. The elucidation of the role that conformational flexibility plays in molecular function will redefine our understanding of the molecular basis of biological function, and the development of appropriate technology to achieve this aim remains one of the major challenges for the future of structural biology. NMR is the technique of choice for studying intrinsically disordered proteins, providing information about structure, flexibility and interactions at atomic resolution even in completely disordered proteins. In particular residual dipolar couplings (RDCs) are sensitive and powerful tools for determining local and long-range structural behaviour in flexible proteins. Here we describe recent applications of the use of RDCs to quantitatively describe the level of local structure in intrinsically disordered proteins involved in replication and transcription in Sendai Virus.
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quantitative conformational analysis of partially folded proteins from residual dipolar couplings application to the molecular recognition element of sendai Virus Nucleoprotein
Journal of the American Chemical Society, 2008Co-Authors: Malene Ringkjobing Jensen, Ewen Lescop, Rob W H Ruigrok, Klaartje Houben, Laurence Blanchard, Martin BlackledgeAbstract:A significant fraction of proteins coded in the human proteome do not fold into stable three-dimensional structures but are either partially or completely unfolded. A key feature of this family of proteins is their proposed capacity to undergo a disorder-to-order transition upon interaction with a physiological partner. The mechanisms governing protein folding upon interaction, in particular the extent to which recognition elements are preconfigured prior to formation of molecular complexes, can prove difficult to resolve in highly flexible systems. Here, we develop a conformational model of this type of protein, using an explicit description of the unfolded state, specifically modified to allow for the presence of transient secondary structure, and combining this with extensive measurement of residual dipolar couplings throughout the chain. This combination of techniques allows us to quantitatively analyze the level and nature of helical sampling present in the interaction site of the partially folded C-terminal domain of Sendai Virus Nucleoprotein (N(TAIL)). Rather than fraying randomly, the molecular recognition element of N(TAIL) preferentially populates three specific overlapping helical conformers, each stabilized by an N-capping interaction. The unfolded strands adjacent to the helix are thereby projected in the direction of the partner protein, identifying a mechanism by which they could achieve nonspecific encounter interactions prior to binding. This study provides experimental evidence for the molecular basis of helix formation in partially folded peptide chains, carrying clear implications for understanding early steps of protein folding.
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structure of recombinant rabies Virus Nucleoprotein rna complex and identification of the phosphoprotein binding site
Journal of Virology, 2001Co-Authors: Guy Schoehn, Danielle Blondel, Frédéric Iseni, Manos Mavrakis, Rob W H RuigrokAbstract:Rabies Virus Nucleoprotein (N) was produced in insect cells, in which it forms Nucleoprotein-RNA (N-RNA) complexes that are biochemically and biophysically indistinguishable from rabies Virus N-RNA. We selected recombinant N-RNA complexes that were bound to short insect cellular RNAs which formed small rings containing 9 to 11 N monomers. We also produced recombinant N-RNA rings and viral N-RNA that were treated with trypsin and that had lost the C-terminal quarter of the Nucleoprotein. Trypsin-treated N-RNA no longer bound to recombinant rabies Virus phosphoprotein (the viral polymerase cofactor), so the presence of the C-terminal part of N is needed for binding of the phosphoprotein. Both intact and trypsin-treated recombinant N-RNA rings were analyzed with cryoelectron microscopy, and three-dimensional models were calculated from single-particle image analysis combined with back projection. Nucleoprotein has a bilobed shape, and each monomer has two sites of interaction with each neighbor. Trypsin treatment cuts off part of one of the lobes without shortening the protein or changing other structural parameters. Using negative-stain electron microscopy, we visualized phosphoprotein bound to the tips of the N-RNA rings, most likely at the site that can be removed by trypsin. Based on the shape of N determined here and on structural parameters derived from electron microscopy on free rabies Virus N-RNA and from nucleocapsid in Virus, we propose a low-resolution model for rabies Virus N-RNA in the Virus.
Jin Wang - One of the best experts on this subject based on the ideXlab platform.
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reply to jensen and blackledge dual quantifications of intrinsically disordered proteins by nmr ensembles and molecular dynamics simulations
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Yong Wang, Sonia Longhi, Philippe Roche, Jin WangAbstract:Jensen and Blackledge (1) compared the residual dipolar couplings (RDCs) of the molecular recognition element of the C-terminal domain of the measles Virus Nucleoprotein (NTAIL) computed from the NMR-based ensembles and the ones computed from the ensemble of our molecular dynamics (MD) simulations (2). They found that the NMR-based ensemble led to better consistency with experimental RDCs than the MD ensemble. However, the MD ensemble did reproduce well the experimental secondary chemical shifts (CSs). They speculated that this may possibly arise from the fact that RDCs are more sensitive to orientational order of local helical structures than CSs. On the basis of this sensitivity, they conclude that RDCs can … [↵][1]1To whom correspondence should be addressed. E-mail: jin.wang.1{at}stonybrook.edu. [1]: #xref-corresp-1-1
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multiscaled exploration of coupled folding and binding of an intrinsically disordered molecular recognition element in measles Virus Nucleoprotein
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Yong Wang, Sonia Longhi, Xiakun Chu, Philippe Roche, Wei Han, Erkang Wang, Jin WangAbstract:Numerous relatively short regions within intrinsically disordered proteins (IDPs) serve as molecular recognition elements (MoREs). They fold into ordered structures upon binding to their partner molecules. Currently, there is still a lack of in-depth understanding of how coupled binding and folding occurs in MoREs. Here, we quantified the unbound ensembles of the α-MoRE within the intrinsically disordered C-terminal domain of the measles Virus Nucleoprotein. We developed a multiscaled approach by combining a physics-based and an atomic hybrid model to decipher the mechanism by which the α-MoRE interacts with the X domain of the measles Virus phosphoprotein. Our multiscaled approach led to remarkable qualitative and quantitative agreements between the theoretical predictions and experimental results (e.g., chemical shifts). We found that the free α-MoRE rapidly interconverts between multiple discrete partially helical conformations and the unfolded state, in accordance with the experimental observations. We quantified the underlying global folding–binding landscape. This leads to a synergistic mechanism in which the recognition event proceeds via (minor) conformational selection, followed by (major) induced folding. We also provided evidence that the α-MoRE is a compact molten globule-like IDP and behaves as a downhill folder in the induced folding process. We further provided a theoretical explanation for the inherent connections between “downhill folding,” “molten globule,” and “intrinsic disorder” in IDP-related systems. Particularly, we proposed that binding and unbinding of IDPs proceed in a stepwise way through a “kinetic divide-and-conquer” strategy that confers them high specificity without high affinity.
Matthias Wolf - One of the best experts on this subject based on the ideXlab platform.
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cryo em structure of the ebola Virus Nucleoprotein rna complex at 3 6 a resolution
Nature, 2018Co-Authors: Takeshi Noda, Yoshihiro Kawaoka, Yukihiko Sugita, Hideyuki Matsunami, Matthias WolfAbstract:Ebola Virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are Viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical Nucleoprotein–RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The Nucleoprotein–RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and Nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola Virus Nucleoprotein–RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola Virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by Nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of Nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions that lead to the formation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development. Near-atomic resolution cryo-electron microscopy structures of the Zaire ebolaVirus Nucleoprotein indicate a complex transition from the RNA-free to RNA-bound forms of the protein, and reveal the mechanism of oligomer formation and helical assembly.
