Baker Max

Barba-Spaeth Giovanna

Blachier Simon

Brelot Anne

Enninga Jost

Groen Johannes

Sartori-Rupp Anna

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13004Multimodal and correlative imaging approaches to study early stages of SARS-CoV 2 infection Dr Johannes Groen1,2, Mr Simon  Blachier2, Dr Max Baker3, Dr Giovanna Barba-Spaeth3, Dr Jost  Enninga2, Dr Anne Brelot2, Dr Anna Sartori-Rupp1 1NanoImaging Core Facility, Institut Pasteur, Paris, France, 2Dynamics in Host Pathogen Interaction, Institut Pasteur, Paris, France, 3FlavImmunity, Institut Pasteur, Paris, France  Background In this work we aim to investigate SARS-CoV-2 entry mechanisms using cutting edge cryo-methodologies to obtain structural insights in the mechanism of infection. SARS-CoV-2 is the causative agent for the recent Covid pandemic and as such has been the centre of both scientific and public attention over the past few years. While this has resulted in numerous publications over a very short amount of time, much is still unknown, also about the entry pathway. For the virus to enter it needs 2 steps, first the viral Spike protein needs to bind to a host cell surface receptor, the most common being ACE-2, although others have been reported to facilitate SARS-CoV-2 entry. Two possible pathways have been identified: direct viral fusion at the cell membrane or viral endocytosis and fusion once internalised. This depends on the presence of host-cell proteases (most commonly TMPRSS2) at the cell surface, as the viral spike protein needs to be cleaved before fusion can commence.   Precisely identifying sites of binding and/or fusion is difficult when doing an infection experiment. Here we aim to use correlative and multimodal imaging in cryogenic conditions to study the viral entry within the very first hour, at a time that the cell-population is basically unaware of an ongoing infection and not releasing stress-effectors. For this we are using cells that can only be infected through the endocytic pathway and a fluorescently labelled virus, which we want to track in a correlative manner. High-resolution fluorescence data is essential to localize the areas of interest and the recent purchase of the cryo-stellaris 8 confocal microscope (Leica) allows us to collect this type of data. After initial mapping and localization of areas of interest, we aim to study the samples using either cryo soft X-ray tomography (cryo-SXT), and/or cryo electron tomography (cryo-ET). Both methods have the advantage of providing 3D information of biological samples preserved as close as possible to their native state. Cryo-SXT is a transmission based, medium-resolution (30 nm half pitch) but high throughput synchrotron technique that relies on the absorption of the X-ray light by the sample. It is a powerful technique to study the cellular ultrastructure of thick samples (<10 micron) and can even be used for quantification by comparing the absorption coefficients of different structures. Cryo-ET is a transmission-based technique that does require very thin samples (<300nm) but can provide nanometre resolution data in return. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/).BIO Web of Conferences 129, 13004 (2024) https://doi.org/10.1051/bioconf/202412913004EMC 2024Using this technique, molecular interactions can be visualized and using sub-tomogram averaging, protein structures can be resolved in situ.  Methods Mammalian cells are cultured on classical TEM Quantifoil grids and infected with SARS-CoV-2 at an MOI of 10. To synchronize the start of the infection, the virus and cells are kept at 4 degrees Celsius, as at this temperature no fusion takes place, and after 30 minutes the cells are moved back to the incubator at 37 °C. The grids are then fixed by plunge-freezing at various timepoints during the first hour of infection. Next, cryo-fluorescence is used to localize sites of interest. The same sample can then be used either for cryo-SXT acquisitions, or for cryo-ET of cell peripheries and of cryo-FIB milled lamellae. Collected tilt series have to be aligned and reconstructed to obtain the final 3D tomograms which can be used for the correlation, segmentation and analysis.  Results By registering the fluorescent map obtained at the confocal microscope with the low magnification TEM map of the grid, we were able to locate areas of interest. Lamellae preparation was required only for the later timepoints as in this timeframe the internalised virus is located in thicker areas of the cells (>300 nm). We visualized the virus at the cell surface and inside vesicles, bound to surface proteins or in the process of fusion. Using cryo-SXT we were able to confirm some of the phenotypes that we observed by cryo-ET, and it allowed us to put it into perspective of a larger area of the cell. It was possible to compare cellular morphologies, and the changes thereof over the course of the first hour of infection.  Conclusion Visualizing different stages of infection within their native environment can greatly improve the understanding of their underlying cellular processes. Here we use 3 complementary modalities that allow us to look at the SARS-CoV-2 infection process with complementary imaging methods at low (cryo-confocal), medium (cryo-SXT) and high (cryo-ET) resolution. While this approach is very time-consuming, it gives a more complete multiscale overview of the complex mechanism of virus-host interaction.  Keywords: cryo-CLEM; CLXM; SARS-CoV-2; Host-Pathogen-Interaction Reference: Bykov, Y. S., Cortese, M., Briggs, J. A. G., & Bartenschlager, R. (2016). Correlative light and electron microscopy methods for the study of virus–cell interactions. In FEBS Letters.  2BIO Web of Conferences 129, 13004 (2024) https://doi.org/10.1051/bioconf/202412913004EMC 2024Collinson, L. M., Domart, M.-C., Carzaniga, R., Razi, M., Guttmann, P., Schneider, G., Pereiro, E., Tooze, S. A., & Duke, E. (2017). Soft X-Ray Tomography: Filling the Gap Between Light and Electrons for Imaging Hydrated Biological Cells. Microscopy and Microanalysis.  Jackson, C. B., Farzan, M., Chen, B., & Choe, H. (2022). Mechanisms of SARS-CoV-2 entry into cells. Nature reviews Molecular cell biology, 23(1), 3-20. Klein, S., Wachsmuth-Melm, M., Winter, S. L., Kolovou, A., & Chlanda, P. (2021). Cryo-correlative light and electron microscopy workflow for cryo-focused ion beam milled adherent cells. In Methods in Cell Biology (Vol. 162).   3BIO Web of Conferences 129, 13004 (2024) https://doi.org/10.1051/bioconf/202412913004EMC 2024

Multimodal and correlative imaging approaches to study early stages of SARS-CoV 2 infection

https://www.bio-conferences.org/articles/bioconf/pdf/2024/48/bioconf_emc2024_13004.pdf

Multimodal and correlative imaging approaches to study early stages of SARS-CoV 2 infection

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