Nucleocapsid self-assembly is an essential yet elusive step in virus replication. Using time-resolved small-angle X-ray scattering on a model icosahedral ssRNA virus, we reveal a previously unreported kinetic pathway. Initially, RNA-bound capsid subunits rapidly accumulate beyond the stoichiometry of native virions. This is followed by a disorder-to-order transition characterized by glass-like relaxation dynamics and the release of excess subunits. Our molecular dynamics simulations, employing a coarse-grained elastic model, confirm the physical feasibility of self-ordering accompanied by subunit release. The relaxation can be modeled by an exponential integral decay on the mean squared radius of gyration, with relaxation times varying within the second range depending on RNA type and subunit concentration. A nanogel model suggests that the initially disordered nucleoprotein complexes quickly reach an equilibrium size, while their mass fractal dimension continues to evolve. Understanding virus self-assembly is not only crucial for combating viral infections, but also for designing synthetic virus-inspired nanocages for drug delivery applications.
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