Self-assembly dynamics of simple icosahedral nucleocapsids under high and low capsid protein concentrations

All viruses on Earth depend on host cell machinery for replication, a process that entails a complex self-assembly mechanism. The cowpea chlorotic mottle virus (CCMV) is a simple, icosahedral, non-enveloped, single-stranded RNA (ssRNA) plant virus that exhibits the remarkable ability to self-assemble in vitro from purified components. I will present the self-assembly dynamics of CCMV, investigated using two complementary techniques: time-resolved small-angle X-ray scattering (TR-SAXS) with synchrotron source and total internal reflection fluorescence microscopy (TIRFM), which probe conditions of high (µM) and low (nM) capsid protein concentrations, respectively. Under high capsid protein concentrations, TR-SAXS reveals that capsid proteins bind to RNA within a hundred of milliseconds, forming nucleoprotein complexes that subsequently undergo a disorder-to-order transition characterized by a glass-like relaxation dynamic over several seconds. During the transition, capsid proteins in excess are released, a feature reproduced by molecular dynamics simulations. Conversely, under low capsid protein concentrations, a single-molecule TIRFM assay in which viral RNA is grafted on a substrate demonstrates that equilibrium is reached only after more than 10 min, with most nucleoprotein complexes remaining incomplete. Furthermore, capsid proteins exhibit dynamic exchange with the surrounding medium, with an estimated mean residence time on RNA of approximately 2 min. Some other single-molecule quantities not accessible to traditional ensemble-averaging techniques will be also presented. Like many other ssRNA viruses, CCMV replicates in the vicinity of the endoplasmic reticulum, namely, in a dense, active milieu where the local concentration of capsid proteins progressively increases as translation proceeds. Understanding the impact of varying capsid protein concentrations as well as of crowding becomes therefore crucial for gaining a comprehensive insight into viral assembly within a native cellular context. REFERENCES T. Bugea et al. (2024). Nano Lett. 24:14821–14828. G. Tresset et al. (2024). J. Phys. Chem. Lett. 15:10210–10218.