An investigation of SARS-CoV-2 assembly at subcellular resolution by the use of fluorescence microscopy
An investigation of SARS-CoV-2 assembly at subcellular resolution by the use of fluorescence microscopy
- Abstract number
- 209
- Presentation Form
- Submitted Talk
- Corresponding Email
- [email protected]
- Session
- Stream 5: Up Close with the Enemy: Imaging Pathogen-host Dynamics
- Authors
- Dr Katharina Scherer (1), Luca Mascheroni (1), Dr George Carnell (1), Lucia Wunderlich (1), Dr Stanislaw Makarchuk (1), Marius Brockhoff (1), Dr Ana Fernandez-Villegas (1), Max Barysevich (1), Dr Hazel Stewart (1), Maria Suau Sans (1), Charlotte George (1), Jacob Lamb (1), Dr Gabriele Kaminski-Schierle (1), Prof Jonathan Heeney (1), Prof Clemens Kaminski (1)
- Affiliations
-
1. University of Cambridge
- Keywords
SARS-CoV-2, COVID, coronavirus, fluorescence, light sheet microscopy, expansion microscopy, cell biology, host cell interaction, infectious disease
- Abstract text
SARS-CoV-2 has recently become the most important coronavirus due to the current COVID-19 global pandemic. Despite being the target of much research effort, relatively little is known about the dynamics of replication within cells. Here, we use light sheet imaging, in combination with expansion microscopy, to study viral replication at sub-cellular detail, beyond the diffraction limit.
Due to the CL3-nature of the virus, live imaging is not possible and so we elected to image fixed cells at various hours afer infection. By first characterising the expression profiles of the four different structural proteins of SARS-CoV-2, we were able to unambiguously assign an infection stage on the single-cell level.
Our experiments show that the nucleoprotein is expressed first and present in vesicle-like structures distinct from the location of the other three structural proteins (spike, membrane and envelope); as the infection progresses, diffused cytosolic signal from the nucleoprotein gradually increases alongside the vesicle formations. We analysed these vesicle-like structures in further detail. We find that the nucleoprotein accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We hope that our continuing experiments will soon shed more light on how the nucleoprotein assembles with the RNA that is replicated inside double-membrane vesicles.