Visualising the self-assembly dynamics of icosahedral viruses through fluorescence microscopy at the single-particle level

All viruses on Earth use the machinery of host cells to replicate themselves. Therefore, there is a lot of ongoing work to understand the dynamics of the self-assembly process that forms viral capsids from proteins and nucleic acids. Recent research using time-resolved small-angle X-ray scattering (TR-SAXS) has provided data with nanometric scale and millisecond resolution on the self-assembly process of icosahedral capsids. Nevertheless, TR-SAXS offers only averaged information on the pathway leading to fully-formed capsids.Our goal is to investigate the self-assembly process of cowpea chlorotic mottle virus (CCMV)—a nonenveloped, icosahedral ssRNA virus—at the single-molecule level with total internal reflection fluorescence microscopy (TIRFM). Recent studies have demonstrated the possibility to monitor in real time the self-assembly process of a complete capsid around genomic RNA immobilized on a glass substrate by interferometric light scattering. In contrast, TIRFM offers higher sensitivity to individual capsid subunits binding/unbinding events on RNA, enabling assembly experiments in crowded macromolecular environments, since only fluorescence is probed. Preliminary results have shown that our setup can observe binding events of red-labelled capsid subunits on various green-labelled genomic RNA segments—transcribed in vitro—which were specifically attached onto a coverslip. Moreover, for a chosen concentration of labelled capsid subunits, we observed a decrease of the assembly time from ten to five minutes as buffer salinity is increased. Currently, we are studying the equilibrium dynamics of binding and unbinding events by using a step-fit algorithm. First results show a mean residence time of capsid subunits onto RNA of 100 s and a constant association of 0.187 s−1. We still need to collect more data to understand how those parameters evolve with the concentration of capsid subunits.