Analyse d'images de microscopie électronique de biopolymères hélicoïdaux flexibles

Flexible helical protein polymers exemplified by actin filaments, microtubules and bacterial flagella areubiquitous in biology. Due to their size and intrinsic irregularities, the structure of these polymers cannot be solved by Xraycrystallography. Since half a century, three-dimensional (3D) reconstruction from two-dimensional (2D) ElectronMicroscopy (EM) images appears as a method of choice to solve the structure of large helical polymers. However,depending on the degree of flexibility of the analyzed helices, the 3D reconstruction process can still be a daunting task.For the most regular helices, the classical reciprocal space-based Fourier-Bessel approach can allow both to determinethe helical symmetry and to calculate 3D structures. For more flexible structures, recent “single-particle” approachesconsist in segmentation of long irregular helices into short (i.e. locally more regular) segments and their processing asasymmetrical objects with defined symmetry-imposed constraints (Egelman, 2000; Sachse et al., 2007). However, twomajor difficulties remain: the heterogeneous data must be sorted into homogeneous populations and the helical symmetryfor each population has to be determined. In the presented work, we explored various single-particle approaches,developed new analysis methods, and implemented most of them into a user-friendly processing pipeline. The targetbiological objects were helical nucleocapsids of two negative strand RNA viruses, Measles (MeV) and VesicularStomatitis Virus (VSV ; the prototype for Rabies), the latter being particularly flexible in terms of helical parameters(diameter, number of subunits per turn). Nucleocapsids are formed by the viral genomic RNA coated by thenucleoprotein and serve as a template for viral replication and transcription. To overcome the heterogeneity problem, weused 2D classification, described general processing protocols and applications for helical segments, and introduced anew classification method based on the power spectra of the images. The determination of helical symmetry(ies) wasaddressed by a novel approach relying on ab initio exhaustive search of helical parameters whereby we start from asingle 2D image, reconstruct as many 3D structures as parameters to test by cropping the image and assigning views tothe obtained segments, and calculate the cross-correlation (CC) of the reprojection of the 3D model with the initialimage. Applied to artificial data sets, the method was effectively able to detect a maximum of CC for the true symmetryparameters, but also showed intrinsic ambiguities of helical symmetry determination on which we extensively comment.Altogether, the result of this method-oriented work allowed us to address several biological questions. First, the 3Dreconstruction by negative stain EM of two forms of nucleocapsids of MeV coupled to a docking of a homologouscrystal structure enabled us to determine the orientation of the nucleoprotein and of the RNA in the nucleocapsids.Secondly, we assessed the structure of in vitro formed nucleocapsids of VSV and showed that assemblies close to thenative viral nucleocapsids can be formed in the absence of any other viral proteins, thus providing new insights into theassembly of this virus. As a perspective of this work, our pipeline of flexible helical analysis is being extended andsuccessfully used for other projects.Le virus de la Rougeole reste le plus meurtrier des virus contre lesquels il existe un vaccin, avec environ 350000 décès par an dans le monde. Ce virus appartient à la famille des Paramyxoviridae, qui sont des virus enveloppés de forme sphérique dont le génome est composé d’un seul brin d’ARN de polarité négative. L’élément central de la réplication et de la transcription du génome viral est le complexe, de forme hélicoïdale, entre l’ARN du virus et la nucléoprotéine. Cette association intime appelée nucléocapside a des propriétés étonnantes non encore élucidées. En effet, l’ARN des virus à ARN négatif a la particularité de n’être jamais nu, même lors des étapes de réplication/transcription nécessitant pourtant le passage de la polymérase virale. On suppose que l’interaction avec la phosphoprotéine, cofacteur de la polymérase, provoque un changement de la conformation de la nucléoprotéine pour rendre l’ARN viral accessible à la polymérase. Lorsque la nucléoprotéine est exprimée dans des cellules d’insectes, elle se fixe aux ARNs cellulaires et forme des nucléocapsides recombinantes. Les études précédentes sur d’autres virus à ARN négatif (Rage, Marbourg, Sendaï) ont montré que les nucléocapsides recombinantes sont semblables aux nucléocapsides virales. Au sein de la nucléocapside, le domaine C-terminal de la nucléoprotéine joue un rôle crucial en interagissant avec de nombreux partenaires viraux et cellulaires, notamment avec la phosphoprotéine dans les étapes de réplication/transcription du génome viral. Cependant, des observations en microscopie électronique à transmission avaient montré que les nucléocapsides recombinantes contenant la nucléoprotéine entière était trop flexibles pour envisager leur reconstruction tridimensionnelle par analyse d’image, ce qui avait conduit à les rigidifier par un traitement protéasique dont l’effet latéral est justement l’élimination du domaine C-terminal de la nucléoprotéine. Nous avons mis au point des conditions de préparation en coloration négative permettant de rigidifier la nucléocapside intacte, afin d’en calculer une reconstruction tridimensionnelle à basse résolution et de la comparer avec celle de la nucléocapside protéolysée. Nous avons ainsi montré que les nucléocapsides de la Rougeole changeaient radicalement de structure tridimensionnelle en réponse au traitement protéolytique, non seulement en terme de pas de l’hélice ou de nombre de sous-unités par tour, mais aussi au niveau de la conformation de la nucléoprotéine et de ses contacts avec les sous-unités adjacentes, ce qui n’avait encore jamais été observé aussi clairement

Analyse d'images de microscopie électronique de biopolymères hélicoïdaux flexibles

Le virus de la Rougeole reste le plus meurtrier des virus contre lesquels il existe un vaccin, avec environ 350000 décès par an dans le monde. Ce virus appartient à la famille des Paramyxoviridae, qui sont des virus enveloppés de forme sphérique dont le génome est composé d un seul brin d ARN de polarité négative. L élément central de la réplication et de la transcription du génome viral est le complexe, de forme hélicoïdale, entre l ARN du virus et la nucléoprotéine. Cette association intime appelée nucléocapside a des propriétés étonnantes non encore élucidées. En effet, l ARN des virus à ARN négatif a la particularité de n être jamais nu, même lors des étapes de réplication/transcription nécessitant pourtant le passage de la polymérase virale. On suppose que l interaction avec la phosphoprotéine, cofacteur de la polymérase, provoque un changement de la conformation de la nucléoprotéine pour rendre l ARN viral accessible à la polymérase. Lorsque la nucléoprotéine est exprimée dans des cellules d insectes, elle se fixe aux ARNs cellulaires et forme des nucléocapsides recombinantes. Les études précédentes sur d autres virus à ARN négatif (Rage, Marbourg, Sendaï) ont montré que les nucléocapsides recombinantes sont semblables aux nucléocapsides virales. Au sein de la nucléocapside, le domaine C-terminal de la nucléoprotéine joue un rôle crucial en interagissant avec de nombreux partenaires viraux et cellulaires, notamment avec la phosphoprotéine dans les étapes de réplication/transcription du génome viral. Cependant, des observations en microscopie électronique à transmission avaient montré que les nucléocapsides recombinantes contenant la nucléoprotéine entière était trop flexibles pour envisager leur reconstruction tridimensionnelle par analyse d image, ce qui avait conduit à les rigidifier par un traitement protéasique dont l effet latéral est justement l élimination du domaine C-terminal de la nucléoprotéine. Nous avons mis au point des conditions de préparation en coloration négative permettant de rigidifier la nucléocapside intacte, afin d en calculer une reconstruction tridimensionnelle à basse résolution et de la comparer avec celle de la nucléocapside protéolysée. Nous avons ainsi montré que les nucléocapsides de la Rougeole changeaient radicalement de structure tridimensionnelle en réponse au traitement protéolytique, non seulement en terme de pas de l hélice ou de nombre de sous-unités par tour, mais aussi au niveau de la conformation de la nucléoprotéine et de ses contacts avec les sous-unités adjacentes, ce qui n avait encore jamais été observé aussi clairement.Flexible helical protein polymers exemplified by actin filaments, microtubules and bacterial flagella areubiquitous in biology. Due to their size and intrinsic irregularities, the structure of these polymers cannot be solved by Xraycrystallography. Since half a century, three-dimensional (3D) reconstruction from two-dimensional (2D) ElectronMicroscopy (EM) images appears as a method of choice to solve the structure of large helical polymers. However,depending on the degree of flexibility of the analyzed helices, the 3D reconstruction process can still be a daunting task.For the most regular helices, the classical reciprocal space-based Fourier-Bessel approach can allow both to determinethe helical symmetry and to calculate 3D structures. For more flexible structures, recent single-particle approachesconsist in segmentation of long irregular helices into short (i.e. locally more regular) segments and their processing asasymmetrical objects with defined symmetry-imposed constraints (Egelman, 2000; Sachse et al., 2007). However, twomajor difficulties remain: the heterogeneous data must be sorted into homogeneous populations and the helical symmetryfor each population has to be determined. In the presented work, we explored various single-particle approaches,developed new analysis methods, and implemented most of them into a user-friendly processing pipeline. The targetbiological objects were helical nucleocapsids of two negative strand RNA viruses, Measles (MeV) and VesicularStomatitis Virus (VSV ; the prototype for Rabies), the latter being particularly flexible in terms of helical parameters(diameter, number of subunits per turn). Nucleocapsids are formed by the viral genomic RNA coated by thenucleoprotein and serve as a template for viral replication and transcription. To overcome the heterogeneity problem, weused 2D classification, described general processing protocols and applications for helical segments, and introduced anew classification method based on the power spectra of the images. The determination of helical symmetry(ies) wasaddressed by a novel approach relying on ab initio exhaustive search of helical parameters whereby we start from asingle 2D image, reconstruct as many 3D structures as parameters to test by cropping the image and assigning views tothe obtained segments, and calculate the cross-correlation (CC) of the reprojection of the 3D model with the initialimage. Applied to artificial data sets, the method was effectively able to detect a maximum of CC for the true symmetryparameters, but also showed intrinsic ambiguities of helical symmetry determination on which we extensively comment.Altogether, the result of this method-oriented work allowed us to address several biological questions. First, the 3Dreconstruction by negative stain EM of two forms of nucleocapsids of MeV coupled to a docking of a homologouscrystal structure enabled us to determine the orientation of the nucleoprotein and of the RNA in the nucleocapsids.Secondly, we assessed the structure of in vitro formed nucleocapsids of VSV and showed that assemblies close to thenative viral nucleocapsids can be formed in the absence of any other viral proteins, thus providing new insights into theassembly of this virus. As a perspective of this work, our pipeline of flexible helical analysis is being extended andsuccessfully used for other projects.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Complete structure of the chemosensory array core signalling unit in an E. coli 1 minicell strain

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array

Analyse d'images de microscopie électronique de biopolymères hélicoïdaux flexibles

Flexible helical protein polymers exemplified by actin filaments, microtubules and bacterial flagella areubiquitous in biology. Due to their size and intrinsic irregularities, the structure of these polymers cannot be solved by Xraycrystallography. Since half a century, three-dimensional (3D) reconstruction from two-dimensional (2D) ElectronMicroscopy (EM) images appears as a method of choice to solve the structure of large helical polymers. However,depending on the degree of flexibility of the analyzed helices, the 3D reconstruction process can still be a daunting task.For the most regular helices, the classical reciprocal space-based Fourier-Bessel approach can allow both to determinethe helical symmetry and to calculate 3D structures. For more flexible structures, recent “single-particle” approachesconsist in segmentation of long irregular helices into short (i.e. locally more regular) segments and their processing asasymmetrical objects with defined symmetry-imposed constraints (Egelman, 2000; Sachse et al., 2007). However, twomajor difficulties remain: the heterogeneous data must be sorted into homogeneous populations and the helical symmetryfor each population has to be determined. In the presented work, we explored various single-particle approaches,developed new analysis methods, and implemented most of them into a user-friendly processing pipeline. The targetbiological objects were helical nucleocapsids of two negative strand RNA viruses, Measles (MeV) and VesicularStomatitis Virus (VSV ; the prototype for Rabies), the latter being particularly flexible in terms of helical parameters(diameter, number of subunits per turn). Nucleocapsids are formed by the viral genomic RNA coated by thenucleoprotein and serve as a template for viral replication and transcription. To overcome the heterogeneity problem, weused 2D classification, described general processing protocols and applications for helical segments, and introduced anew classification method based on the power spectra of the images. The determination of helical symmetry(ies) wasaddressed by a novel approach relying on ab initio exhaustive search of helical parameters whereby we start from asingle 2D image, reconstruct as many 3D structures as parameters to test by cropping the image and assigning views tothe obtained segments, and calculate the cross-correlation (CC) of the reprojection of the 3D model with the initialimage. Applied to artificial data sets, the method was effectively able to detect a maximum of CC for the true symmetryparameters, but also showed intrinsic ambiguities of helical symmetry determination on which we extensively comment.Altogether, the result of this method-oriented work allowed us to address several biological questions. First, the 3Dreconstruction by negative stain EM of two forms of nucleocapsids of MeV coupled to a docking of a homologouscrystal structure enabled us to determine the orientation of the nucleoprotein and of the RNA in the nucleocapsids.Secondly, we assessed the structure of in vitro formed nucleocapsids of VSV and showed that assemblies close to thenative viral nucleocapsids can be formed in the absence of any other viral proteins, thus providing new insights into theassembly of this virus. As a perspective of this work, our pipeline of flexible helical analysis is being extended andsuccessfully used for other projects.Le virus de la Rougeole reste le plus meurtrier des virus contre lesquels il existe un vaccin, avec environ 350000 décès par an dans le monde. Ce virus appartient à la famille des Paramyxoviridae, qui sont des virus enveloppés de forme sphérique dont le génome est composé d’un seul brin d’ARN de polarité négative. L’élément central de la réplication et de la transcription du génome viral est le complexe, de forme hélicoïdale, entre l’ARN du virus et la nucléoprotéine. Cette association intime appelée nucléocapside a des propriétés étonnantes non encore élucidées. En effet, l’ARN des virus à ARN négatif a la particularité de n’être jamais nu, même lors des étapes de réplication/transcription nécessitant pourtant le passage de la polymérase virale. On suppose que l’interaction avec la phosphoprotéine, cofacteur de la polymérase, provoque un changement de la conformation de la nucléoprotéine pour rendre l’ARN viral accessible à la polymérase. Lorsque la nucléoprotéine est exprimée dans des cellules d’insectes, elle se fixe aux ARNs cellulaires et forme des nucléocapsides recombinantes. Les études précédentes sur d’autres virus à ARN négatif (Rage, Marbourg, Sendaï) ont montré que les nucléocapsides recombinantes sont semblables aux nucléocapsides virales. Au sein de la nucléocapside, le domaine C-terminal de la nucléoprotéine joue un rôle crucial en interagissant avec de nombreux partenaires viraux et cellulaires, notamment avec la phosphoprotéine dans les étapes de réplication/transcription du génome viral. Cependant, des observations en microscopie électronique à transmission avaient montré que les nucléocapsides recombinantes contenant la nucléoprotéine entière était trop flexibles pour envisager leur reconstruction tridimensionnelle par analyse d’image, ce qui avait conduit à les rigidifier par un traitement protéasique dont l’effet latéral est justement l’élimination du domaine C-terminal de la nucléoprotéine. Nous avons mis au point des conditions de préparation en coloration négative permettant de rigidifier la nucléocapside intacte, afin d’en calculer une reconstruction tridimensionnelle à basse résolution et de la comparer avec celle de la nucléocapside protéolysée. Nous avons ainsi montré que les nucléocapsides de la Rougeole changeaient radicalement de structure tridimensionnelle en réponse au traitement protéolytique, non seulement en terme de pas de l’hélice ou de nombre de sous-unités par tour, mais aussi au niveau de la conformation de la nucléoprotéine et de ses contacts avec les sous-unités adjacentes, ce qui n’avait encore jamais été observé aussi clairement

Analysis of electron microscopy images of flexible helical bio-polymers

Le virus de la Rougeole reste le plus meurtrier des virus contre lesquels il existe un vaccin, avec environ 350000 décès par an dans le monde. Ce virus appartient à la famille des Paramyxoviridae, qui sont des virus enveloppés de forme sphérique dont le génome est composé d’un seul brin d’ARN de polarité négative. L’élément central de la réplication et de la transcription du génome viral est le complexe, de forme hélicoïdale, entre l’ARN du virus et la nucléoprotéine. Cette association intime appelée nucléocapside a des propriétés étonnantes non encore élucidées. En effet, l’ARN des virus à ARN négatif a la particularité de n’être jamais nu, même lors des étapes de réplication/transcription nécessitant pourtant le passage de la polymérase virale. On suppose que l’interaction avec la phosphoprotéine, cofacteur de la polymérase, provoque un changement de la conformation de la nucléoprotéine pour rendre l’ARN viral accessible à la polymérase. Lorsque la nucléoprotéine est exprimée dans des cellules d’insectes, elle se fixe aux ARNs cellulaires et forme des nucléocapsides recombinantes. Les études précédentes sur d’autres virus à ARN négatif (Rage, Marbourg, Sendaï) ont montré que les nucléocapsides recombinantes sont semblables aux nucléocapsides virales. Au sein de la nucléocapside, le domaine C-terminal de la nucléoprotéine joue un rôle crucial en interagissant avec de nombreux partenaires viraux et cellulaires, notamment avec la phosphoprotéine dans les étapes de réplication/transcription du génome viral. Cependant, des observations en microscopie électronique à transmission avaient montré que les nucléocapsides recombinantes contenant la nucléoprotéine entière était trop flexibles pour envisager leur reconstruction tridimensionnelle par analyse d’image, ce qui avait conduit à les rigidifier par un traitement protéasique dont l’effet latéral est justement l’élimination du domaine C-terminal de la nucléoprotéine. Nous avons mis au point des conditions de préparation en coloration négative permettant de rigidifier la nucléocapside intacte, afin d’en calculer une reconstruction tridimensionnelle à basse résolution et de la comparer avec celle de la nucléocapside protéolysée. Nous avons ainsi montré que les nucléocapsides de la Rougeole changeaient radicalement de structure tridimensionnelle en réponse au traitement protéolytique, non seulement en terme de pas de l’hélice ou de nombre de sous-unités par tour, mais aussi au niveau de la conformation de la nucléoprotéine et de ses contacts avec les sous-unités adjacentes, ce qui n’avait encore jamais été observé aussi clairement.Flexible helical protein polymers exemplified by actin filaments, microtubules and bacterial flagella areubiquitous in biology. Due to their size and intrinsic irregularities, the structure of these polymers cannot be solved by Xraycrystallography. Since half a century, three-dimensional (3D) reconstruction from two-dimensional (2D) ElectronMicroscopy (EM) images appears as a method of choice to solve the structure of large helical polymers. However,depending on the degree of flexibility of the analyzed helices, the 3D reconstruction process can still be a daunting task.For the most regular helices, the classical reciprocal space-based Fourier-Bessel approach can allow both to determinethe helical symmetry and to calculate 3D structures. For more flexible structures, recent “single-particle” approachesconsist in segmentation of long irregular helices into short (i.e. locally more regular) segments and their processing asasymmetrical objects with defined symmetry-imposed constraints (Egelman, 2000; Sachse et al., 2007). However, twomajor difficulties remain: the heterogeneous data must be sorted into homogeneous populations and the helical symmetryfor each population has to be determined. In the presented work, we explored various single-particle approaches,developed new analysis methods, and implemented most of them into a user-friendly processing pipeline. The targetbiological objects were helical nucleocapsids of two negative strand RNA viruses, Measles (MeV) and VesicularStomatitis Virus (VSV ; the prototype for Rabies), the latter being particularly flexible in terms of helical parameters(diameter, number of subunits per turn). Nucleocapsids are formed by the viral genomic RNA coated by thenucleoprotein and serve as a template for viral replication and transcription. To overcome the heterogeneity problem, weused 2D classification, described general processing protocols and applications for helical segments, and introduced anew classification method based on the power spectra of the images. The determination of helical symmetry(ies) wasaddressed by a novel approach relying on ab initio exhaustive search of helical parameters whereby we start from asingle 2D image, reconstruct as many 3D structures as parameters to test by cropping the image and assigning views tothe obtained segments, and calculate the cross-correlation (CC) of the reprojection of the 3D model with the initialimage. Applied to artificial data sets, the method was effectively able to detect a maximum of CC for the true symmetryparameters, but also showed intrinsic ambiguities of helical symmetry determination on which we extensively comment.Altogether, the result of this method-oriented work allowed us to address several biological questions. First, the 3Dreconstruction by negative stain EM of two forms of nucleocapsids of MeV coupled to a docking of a homologouscrystal structure enabled us to determine the orientation of the nucleoprotein and of the RNA in the nucleocapsids.Secondly, we assessed the structure of in vitro formed nucleocapsids of VSV and showed that assemblies close to thenative viral nucleocapsids can be formed in the absence of any other viral proteins, thus providing new insights into theassembly of this virus. As a perspective of this work, our pipeline of flexible helical analysis is being extended andsuccessfully used for other projects

SPRING - an image processing package for single-particle based helical reconstruction from electron cryomicrographs.

International audienceHelical reconstruction from electron cryomicrographs has become a routine technique for macromolecular structure determination of helical assemblies since the first days of Fourier-based three-dimensional image reconstruction. In the past decade, the single-particle technique has had an important impact on the advancement of helical reconstruction. Here, we present the software package SPRING that combines Fourier based symmetry analysis and real-space helical processing into a single workflow. One of the most time-consuming steps in helical reconstruction is the determination of the initial symmetry parameters. First, we propose a class-based helical reconstruction approach that enables the simultaneous exploration and evaluation of many symmetry combinations at low resolution. Second, multiple symmetry solutions can be further assessed and refined by single-particle based helical reconstruction using the correlation of simulated and experimental power spectra. Finally, the 3D structure can be determined to high resolution. In order to validate the procedure, we use the reference specimen Tobacco Mosaic Virus (TMV). After refinement of the helical symmetry, a total of 50,000 asymmetric units from two micrographs are sufficient to reconstruct a subnanometer 3D structure of TMV at 6.4Å resolution. Furthermore, we introduce the individual programs of the software and discuss enhancements of the helical reconstruction workflow. Thanks to its user-friendly interface and documentation, SPRING can be utilized by the novice as well as the expert user. In addition to the study of well-ordered helical structures, the development of a streamlined workflow for single-particle based helical reconstruction opens new possibilities to analyze specimens that are heterogeneous in symmetries

Structural Model Of The Tubular Assembly Of The Rous Sarcoma Virus Capsid Protein

The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and Cterminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solidstate NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 310 helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of this MDFF model with an earlier-derived crystallographic model for the planar assembly, the induction of curvature into the RSV CA hexamer lattice arises predominantly from reconfiguration of the NTD-CTD and CTD trimer interfaces. The CTD dimer and CTD trimer interfaces are also intrinsically variable. Hence, deformation of the CA hexamer lattice results from the variable displacement of the CTDs that surround each hexameric turret. Pervasive H-bonding is found at all interdomain interfaces, which may contribute to their malleability. Finally, we find helices at the interfaces of HIV and RSV CA assemblies have very different contact angles, which may reflect differences in the capsid assembly pathway for these viruses

Nucleoprotein-RNA Orientation in the Measles Virus Nucleocapsid by Three-Dimensional Electron Microscopy▿ †

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

Structural landscape of the respiratory syncytial virus nucleocapsids

Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation

Structural virology. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid.

International audienceMeasles 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