Thomas BUGEA has defended his PhD thesis entitled Visualising the self-assembly mechanisms of icosahedral viruses through fluorescence microscopy at the single molecule level under the supervision of Karen PERRONET and Guillaume TRESSET on September 30, 2024.
The diversity of viruses we are facing dictated us to learn how to live with them. To facilitate this coexistence and, above all, combat their proliferation, it has been necessary to understand how they work. This need has led to numerous studies on viruses of various sizes and compositions. Up to now, the results obtained through ensemble measurements contribute to improving our knowledge upon assembly pathways of viral capsids and their dependence on salt and pH. Indeed, most of the techniques used give average results for an entire population, without being able to access to the inherent heterogeneity of the assembly mechanism. Total internal reflection fluorescence microscopy (TIRFM) allows us to work not only at the level of the individual capsid, but also at the level of the individual subunit. This experimental setup has been used on the Cowpea Chlorotic Mottle Virus (CCMV), whose dimers and single-stranded RNA are labelled. Observing the self-assembly mechanism of this icosahedral virus on a coverslip was achieved after a long optimisation stage of the glass passivation method. The surface chemistry we chose, and the several characterisation steps showed that the fluorescent signals collected corresponded exactly to the specific interactions between labelled subunits and single-stranded RNA. Therefore, these experiments combined to the step-fit algorithm Ruptures enabled us to follow the self-assembly dynamics of CCMV at equilibrium. We accessed to information such as the mean number of capsid subunits per RNA and the time between two consecutive events. These initial experiments showed, as expected, an increase in particle size and a decrease in binding rate as the subunit concentration increased. These results, combined with those obtained by mass photometry, indicated that the self-assembly mechanism followed through fluorescence microscopy could be close to give fully formed capsids. Finally, non-equilibrium experiments seemed to suggest that the early steps of assembly were faster when the salt concentration was closer to physiological conditions.