Viral Amyloids Across Scales
The Role of Viral Amyloids in Infection and Neurodegeneration
Amyloids, long regarded as disease-linked aggregates, serve functional roles in biology, including viruses, yet molecular insights remain limited. In this project, I lays the foundation to decipher the viral-host amyloid crosstalk by characterizing amyloid-prone targets across viral families using structural, biochemical, and cellular approaches. Focusing on neurotropic viruses, I assess whether viral targets cross-aggregate tau/Aβ and trigger neuronal stress, offering mechanistic evidence for the neuroinfection-neurodegeneration link and guiding therapies against infection and neurodegeneration. Currently, I have several fibrillating target structures of different viruses which are in the in vitro characterization stage, while having set-up the conception of the in cellulo characterization.
Important Previous Projects
Chikungunya Viral Replication
NsP3 of the emerging chikungunya virus is part of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and located in large cytoplasmic aggregates. I solved the 2.35 Å resolution tubular structure made by a helical arrangement of its alphavirus unique domain (AUD). This helical symmetry is consistent with the crown structure of the membrane-bound mature vRC. The large cytoplasmic aggregates contain viral genomic RNA and capsid as well as host factors required for productive infection. Our results reveal an unexpected role of nsP3 required for productive alphavirus replication or transcription and imply nsP3 as potent antiviral target. NsP1 is a core unit of the vRC crown and responsible for RNA capping and gating by assembling into membrane-associated dodecameric pores. By solving structural snapshots of intermediate states, we provide a mechanistic understanding of the capping pathway, beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate vRNA. We demonstrate specificity for the RNA substrate and show the reversibility of this capping reaction. My critical role in both structural works (Nsp1/3) in solving and interpreting structures and developing the projects resulted in shared first authorship of both collaborative projects.
Kril, V., Hons M. et al. Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture. Nat Commun 15, 8106 (2024).
Jones, R., Hons M. et al. Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores. Proc Natl Acad Sci U S A 120, e2213934120 (2023).
NsP3 of the emerging chikungunya virus is part of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and located in large cytoplasmic aggregates. I solved the 2.35 Å resolution tubular structure made by a helical arrangement of its alphavirus unique domain (AUD). This helical symmetry is consistent with the crown structure of the membrane-bound mature vRC. The large cytoplasmic aggregates contain viral genomic RNA and capsid as well as host factors required for productive infection. Our results reveal an unexpected role of nsP3 required for productive alphavirus replication or transcription and imply nsP3 as potent antiviral target. NsP1 is a core unit of the vRC crown and responsible for RNA capping and gating by assembling into membrane-associated dodecameric pores. By solving structural snapshots of intermediate states, we provide a mechanistic understanding of the capping pathway, beginning with the N7 methylation of a GTP molecule, followed by the covalent linkage of a m7GMP to a conserved histidine in nsP1 and the transfer of this cap structure to a diphosphate vRNA. We demonstrate specificity for the RNA substrate and show the reversibility of this capping reaction. My critical role in both structural works (Nsp1/3) in solving and interpreting structures and developing the projects resulted in shared first authorship of both collaborative projects.
Kril, V., Hons M. et al. Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture. Nat Commun 15, 8106 (2024).
Jones, R., Hons M. et al. Structural basis and dynamics of Chikungunya alphavirus RNA capping by nsP1 capping pores. Proc Natl Acad Sci U S A 120, e2213934120 (2023).
Axon Fragmentation
Human SARM1 is an executioner of axonal fragmentation (Wallerian Degeneration, WD) followed by various axon damage. This process is initiated the depletion of the key cellular metabolite NAD+ in response to axon damage and accelerated by SARM1 NADase activity. We solved a cryo-EM structure SARM1 at 2.7 Å resolution, showing an octameric tightly-packed SARM1 state that avoids premature NADase activity. Inactivation is based on allosteric substrate inhibition by the substrate NAD+. In our model, SARM1 orchestrates energetic catastrophe and cell death in a NAD+ concentration-dependent manner. In two following works, I was involved in project development legitimating my co-corresponding role in these highly collaborative projects. We identified a novel SARM1 inhibitor that stabilizes both the inactive octamer and a newly discovered auto-inhibited duplex. Furthermore, we studied the evolutionary related ortholog ceTIR1 (C. elegans) to understand why WD does not manifest in C. elegans. Our findings imply an evolutionary functional shift from promoting axon preservation in C. elegans to mediating axon degeneration under stress conditions in higher organisms. We determined the structure of ceTIR-1, investigated its enzymatic NADase activity and its effects on cell viability. In distinction to SARM1 octamers, ceTIR-1 can form a readily active 9-mer, in which eight TIR domains are poised for catalytic activity and one is inhibited. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker than that of SARM1 and falls short of consuming the entire cellular NAD+ stock when fully activated.
Sporny, M. et al. Structural basis for SARM1 inhibition and activation under energetic stress. eLife 9, e62021 (2020).
Khazma, T. et al. A duplex structure of SARM1 octamers stabilized by a new inhibitor. Cell. Mol. Life Sci. 80, 16 (2022).
Khazma, T. et al. Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans. Cell Reports 42, (2023).
Human SARM1 is an executioner of axonal fragmentation (Wallerian Degeneration, WD) followed by various axon damage. This process is initiated the depletion of the key cellular metabolite NAD+ in response to axon damage and accelerated by SARM1 NADase activity. We solved a cryo-EM structure SARM1 at 2.7 Å resolution, showing an octameric tightly-packed SARM1 state that avoids premature NADase activity. Inactivation is based on allosteric substrate inhibition by the substrate NAD+. In our model, SARM1 orchestrates energetic catastrophe and cell death in a NAD+ concentration-dependent manner. In two following works, I was involved in project development legitimating my co-corresponding role in these highly collaborative projects. We identified a novel SARM1 inhibitor that stabilizes both the inactive octamer and a newly discovered auto-inhibited duplex. Furthermore, we studied the evolutionary related ortholog ceTIR1 (C. elegans) to understand why WD does not manifest in C. elegans. Our findings imply an evolutionary functional shift from promoting axon preservation in C. elegans to mediating axon degeneration under stress conditions in higher organisms. We determined the structure of ceTIR-1, investigated its enzymatic NADase activity and its effects on cell viability. In distinction to SARM1 octamers, ceTIR-1 can form a readily active 9-mer, in which eight TIR domains are poised for catalytic activity and one is inhibited. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker than that of SARM1 and falls short of consuming the entire cellular NAD+ stock when fully activated.
Sporny, M. et al. Structural basis for SARM1 inhibition and activation under energetic stress. eLife 9, e62021 (2020).
Khazma, T. et al. A duplex structure of SARM1 octamers stabilized by a new inhibitor. Cell. Mol. Life Sci. 80, 16 (2022).
Khazma, T. et al. Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans. Cell Reports 42, (2023).
Cryo-EM Facilities & EM Method Development
I established and operated the EMBL Grenoble cryo-EM facility and the ESRF CM01 beamline, the latter in collaboration with ESRF and IBS staff scientists (Eaazhisai Kandiah, Gregory Effantin). This involved the setup and maintenance of high-end cryo-EM microscopes (Titan Krios G3, Glacios), real-time image processing pipelines involving Scipion and WARP and method development in grid preparation. Cryo-EM grid preparation is limited by manual throughput and poor reproducibility. At EMBL, we developed EasyGrid, a versatile tool for tailored and high-throughput grid preparation. By combining microfluidic dispensing, blot-less control of sample thickness and jet-based vitrification in one framework, Easygrid accelerates the traditionally slow sample optimization procedure in single-particle EM and cell vitrification. Using interferometry to control grid quality on-the-fly, cryo-EM grid quality control becomes automated. EasyGrid allowed the determining novel structures at near-atomic resolution and full vitrification of mammalian cells cultured on grids for cryo-tomography. Our reproducible sample preparation is the basis for further workflows including time-resolved cryo-EM and high-throughput cryo-tomography. I contributed in developing and advancing the idea and rigorous testing of conditions for process optimization.
https://www.esrf.fr/CM01
https://www.embl.org/services-facilities/grenoble/cryoem/
Kandiah, E. et al. CM01: a facility for cryo-electron microscopy at the European Synchrotron. Acta crystallographica. Section D, Structural biology 2019, 75 (Pt 6), 528-535.
Gemin, O. et al. EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control. 2024.01.18.576170 Preprint at https://doi.org/10.1101/2024.01.18.576170 (2024).
I established and operated the EMBL Grenoble cryo-EM facility and the ESRF CM01 beamline, the latter in collaboration with ESRF and IBS staff scientists (Eaazhisai Kandiah, Gregory Effantin). This involved the setup and maintenance of high-end cryo-EM microscopes (Titan Krios G3, Glacios), real-time image processing pipelines involving Scipion and WARP and method development in grid preparation. Cryo-EM grid preparation is limited by manual throughput and poor reproducibility. At EMBL, we developed EasyGrid, a versatile tool for tailored and high-throughput grid preparation. By combining microfluidic dispensing, blot-less control of sample thickness and jet-based vitrification in one framework, Easygrid accelerates the traditionally slow sample optimization procedure in single-particle EM and cell vitrification. Using interferometry to control grid quality on-the-fly, cryo-EM grid quality control becomes automated. EasyGrid allowed the determining novel structures at near-atomic resolution and full vitrification of mammalian cells cultured on grids for cryo-tomography. Our reproducible sample preparation is the basis for further workflows including time-resolved cryo-EM and high-throughput cryo-tomography. I contributed in developing and advancing the idea and rigorous testing of conditions for process optimization.
https://www.esrf.fr/CM01
https://www.embl.org/services-facilities/grenoble/cryoem/
Kandiah, E. et al. CM01: a facility for cryo-electron microscopy at the European Synchrotron. Acta crystallographica. Section D, Structural biology 2019, 75 (Pt 6), 528-535.
Gemin, O. et al. EasyGrid: A versatile platform for automated cryo-EM sample preparation and quality control. 2024.01.18.576170 Preprint at https://doi.org/10.1101/2024.01.18.576170 (2024).
Cohesin in Sister Chromatid Cohesion
My doctoral work focused on the macromolecular complexes of cohesin bound to an interaction partner. Our Nature Communications paper in 2016 described the architecture of engineered human cohesin bound to Pds5B. The HEAT-repeat protein Pds5B forms a curved structure around the NBD of Smc1/Smc3 and bridges the Smc3-Scc1 and SA1-Scc1 interfaces.
Hons, M., Huis In 't Veld, P. J. et al. Topology and structure of an engineered human cohesin complex bound to Pds5B. Nat Commun 2016, 7, 12523.
My doctoral work focused on the macromolecular complexes of cohesin bound to an interaction partner. Our Nature Communications paper in 2016 described the architecture of engineered human cohesin bound to Pds5B. The HEAT-repeat protein Pds5B forms a curved structure around the NBD of Smc1/Smc3 and bridges the Smc3-Scc1 and SA1-Scc1 interfaces.
Hons, M., Huis In 't Veld, P. J. et al. Topology and structure of an engineered human cohesin complex bound to Pds5B. Nat Commun 2016, 7, 12523.