Maturation de la capside du bactériophage T5 : étude structurale et fonctionnelle de la protéine de décoration pb10

Bacteriophage T5, a lytic phage which infects the bacterium Escherichia coli, is composed of an icosahedral capsid containing a double stranded DNA and a tail responsible for DNA transfer into the host cell cytoplasm. The capsid and the tail are assembled separately and then connected to form infectious viruses. The T5 capsid is first assembled as an empty procapsid composed of 775 copies of the major head protein organized in hexamers on the faces and pentamers on the vertices of the icosahedron. A single vertex is occupied by the portal protein which forms an entry channel for DNA. DNA is packaged through the portal channel by a molecular motor thus triggering procapsid expansion. The DNA-filled capsid is then decorated by the protein pb10 which binds on the outer surface in the center of each hexamer, and closed by the connector protein p144 needed for tail attachment.The goals of my PhD were to characterize the structure, function and mechanisms of capsid binding of both pb10 and p144 proteins. I have produced and purified the head completion protein p144 for future exploration of its structure and capsid binding properties. I have solved the solution structure of the decoration protein pb10 by nuclear magnetic resonance (NMR), thus unravelling that pb10 has two domains. The alpha-helical N-terminal domain binds to the capsid and the immunoglobulin-like C-terminal domain is exposed to the environment. Surface plasmon resonance (SPR) showed that that pb10 attachment to the T5 capsid is quasi-irreversible with a picomolar affinity. Site-directed mutagenesis of the binding domain of pb10 suggested that the decoration mechanism is driven by both hydrophobic and electrostatic interactions. Differential scanning calorimetry (DSC) and fluorescence thermal shift assays (FTSA) demonstrated the role of the decoration protein in capsid stabilization. I have also demonstrated that a large protein can be fused to the C-terminal end of pb10 without affecting its high affinity for the T5 capsid. Capsids can thus be functionalized with a protein of interest fused to pb10. Possible applications of this work include antigen presentation for new vaccines.Le bactériophage T5, qui infecte la bactérie Escherichia coli, est constitué d’une capside icosaédrique renfermant un ADN double brin et d’une queue permettant le transfert de son ADN dans le cytoplasme de la cellule hôte. Au cours de la morphogénèse, la capside et la queue sont assemblées séparément puis connectées pour former les particules infectieuses. La capside de T5 est d'abord assemblée sous forme d'une procapside vide constituée de 775 copies d’une protéine unique qui s'organise en hexamères et pentamères pour former respectivement les faces et les sommets de l’icosaèdre. Un des sommets est occupé par la protéine portale qui constitue un pore d’entrée pour l’ADN. L’encapsidation du génome à travers le canal portal, assurée par un moteur moléculaire, provoque l’expansion de la procapside. La capside pleine d'ADN est ensuite décorée par la protéine pb10 qui se fixe sur sa surface externe, au centre des 120 hexamères, et elle est fermée par la protéine p144 qui constitue le point d'ancrage pour la queue.Les objectifs de ma thèse étaient de déterminer la structure, la fonction et le mécanisme de fixation à la capside des deux protéines pb10 et p144. J’ai produit et purifié la protéine de fermeture p144 dont la structure et les caractéristiques de fixation sur la capside seront prochainement étudiées. J’ai résolu la structure de la protéine de décoration pb10 en solution par résonance magnétique nucléaire (RMN), ce qui a révélé une protéine formée de deux domaines. Le domaine N-terminal structuré en hélices alpha se fixe à la capside, alors que le domaine C-terminal, de type immunoglobuline, est exposé au solvant. La détermination des constantes cinétiques d'association et de dissociation par résonance plasmonique de surface (SPR) a permis de montrer que pb10 se fixe sur la capside de T5 de manière quasi-irréversible, avec une affinité de l’ordre du picomolaire. Des expériences de mutagenèse dirigée sur le domaine de fixation de pb10 indiquent que cette forte affinité repose sur un réseau d’interactions électrostatiques et hydrophobes. J’ai démontré que pb10 participe à la stabilisation de la capside de T5 par des techniques de calorimétrie différentielle à balayage (DSC) et thermo-fluorescence (FTSA). Par ailleurs, j'ai démontré qu’il était possible de fusionner des protéines de grande taille au domaine C-terminal de pb10 sans affecter sa haute affinité pour la capside. J’ai ainsi pu fonctionnaliser des capsides avec une protéine d’intérêt fusionnée à pb10. Ces résultats ouvrent la voie vers diverses applications, dont la présentation d’antigènes en vue de créer de nouveaux vaccins

Charge-independent mass spectrometry of single virus capsids above 100MDa with nanomechanical resonators

Most technologies, including conventional mass spectrometry, struggle to measure the mass of particles in the MDa to GDa range. Although this mass range appears optimal for nanomechanical resonators, early nanomechanical-MS systems suffered from prohibitive sample loss, extended analysis time or inadequate resolution. Here, we report on a novel system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30 MDa polystyrene nanoparticles with a detection efficiency 6 orders of magnitude higher than previous nanomechanical-MS systems with ion guides, and successfully performed the highest molecular mass measurement to date with less than 1 picomole of bacteriophage T5 105 MDa viral capsids

Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

International audienceThe promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here, we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunization with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8 + T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes a unique DNA-free bacteriophage capsid able to display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform

Neutral mass spectrometry of virus capsids above 100 megadaltons with nanomechanical resonators

International audienceMeasurement of the mass of particles in the mega- to gigadalton range is challenging with conventional mass spectrometry. Although this mass range appears optimal for nanomechanical resonators, nanomechanical mass spectrometers often suffer from prohibitive sample loss, extended analysis time, or inadequate resolution. We report on a system architecture combining nebulization of the analytes from solution, their efficient transfer and focusing without relying on electromagnetic fields, and the mass measurements of individual particles using nanomechanical resonator arrays. This system determined the mass distribution of ~30-megadalton polystyrene nanoparticles with high detection efficiency and effectively performed molecular mass measurements of empty or DNA-filled bacteriophage T5 capsids with masses up to 105 megadaltons using less than 1 picomole of sample and with an instrument resolution above 100

Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design

Abstract The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunisation with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8 + T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes the first DNA-free bacteriophage capsid able to irreversibly display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform

High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

International audienceBacteriophage capsids constitute icosahedral shells of exceptional stability that protect the viral genome. Many capsids display on their surface decoration proteins whose structure and function remain largely unknown. The decoration protein pb10 of phage T5 binds at the centre of the 120 hexamers formed by the major capsid protein. Here we determined the 3D structure of pb10 and investigated its capsid-binding properties using NMR, SAXS, cryoEM and SPR. Pb10 consists of an α-helical capsid-binding domain and an Ig-Like domain exposed to the solvent. It binds to the T5 capsid with a remarkably high affinity and its binding kinetics is characterized by a very slow dissociation rate. We propose that the conformational exchange events observed in the capsid-binding domain enable rearrangements upon binding that contribute to the quasi-irreversibility of the pb10-Capsid interaction. Moreover we show that pb10 binding is a highly cooperative process, which favours immediate rebinding of newly dissociated pb10 to the 120 hexamers of the capsid protein. In extreme conditions, pb10 protects the phage from releasing its genome. We conclude that pb10 may function to reinforce the capsid thus favouring phage survival in harsh environments

Erratum: High affinity anchoring of the decoration protein pb10 onto the bacteriophage T5 capsid

Todos sabemos que cuando volvemos a leer un libro, no retornamos del mismo modo. Porque entre aquellas lecturas y estas pasó tiempo. Tiempo histórico. Como escribió el historiador Marc Bloch, continuidad, pero también cambio perpetuo. Por eso mismo, afortunadamente, el regreso hacia las páginas de este libro ha crecido, ha madurado y ha sobrevivido, como yo. Y como el aula.Facultad de Humanidades y Ciencias de la Educació