VIRAL-MAP: Viral Infection and Amyloid-Linked Mechanism Across Pathology
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. This programme establishes the first comprehensive structural and functional framework for viral amyloids, focusing on neurotropic and neuroinvasive viruses. Functional and pathological amyloid states can both engage nucleic acids— a principle that extends to virus-derived peptides that co-assemble with nucleic acids to drive innate immune reaction. I assess whether viral amyloid interact with nucleic acids, lipids, and host proteins — molecular interactions that collectively drive neuronal stress and innate immune activation as downstream consequences — or targets cross-seed tau/Aβ aggregation. This strategy may offer mechanistic evidence for the neuroinfection-neurodegeneration link and and guiding the development of therapies against infection and neurodegeneration. Currently, I have several fibrillating target structures of different viruses which are in structural evaluation, while having established the in cellulo characterization framework . Functional studies in the third column deliver a drug design entry points.
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 contributions to both structural works (NsP1/3) — solving and interpreting structures and driving project development — 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 contributions to both structural works (NsP1/3) — solving and interpreting structures and driving project development — 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 justifying my co-corresponding author role in these highly collaborative projects. In a structure-guided drug design study we tested several compounds in a DRG axon degeneration assay and a novel SARM1 inhibitor and auto-inhibited duplex state were subsequently characterised. 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 is insufficient to deplete the entire cellular NAD+ pool upon full activation.
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 justifying my co-corresponding author role in these highly collaborative projects. In a structure-guided drug design study we tested several compounds in a DRG axon degeneration assay and a novel SARM1 inhibitor and auto-inhibited duplex state were subsequently characterised. 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 is insufficient to deplete the entire cellular NAD+ pool upon full activation.
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 to developing and advancing the concept, and to rigorously testing conditions for process optimization.
Previous cryo-EM Facilities:
CM01 beamline
Cyro-EM EMBL Grenoble
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 to developing and advancing the concept, and to rigorously testing conditions for process optimization.
Previous cryo-EM Facilities:
CM01 beamline
Cyro-EM EMBL Grenoble
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.