  The formation of a viral capsid -- the highly-ordered protein shell that
surrounds the genome of a virus -- is the canonical example of self-assembly.
The capsids of many positive-sense RNA viruses spontaneously assemble from in
vitro mixtures of the coat protein and RNA. The high yield of proper capsids
that assemble is remarkable, given their structural complexity: 180 identical
proteins must arrange into three distinct local configurations to form an
icosahedral capsid with a triangulation number of 3 (T = 3). Despite a wealth
of data from structural studies and simulations, even the most fundamental
questions about how these structures assemble remain unresolved. Experiments
have not determined whether the assembly pathway involves aggregation or
nucleation, or how the RNA controls the process. Here we use interferometric
scattering microscopy to directly observe the in vitro assembly kinetics of
individual, unlabeled capsids of bacteriophage MS2. By measuring how many coat
proteins bind to individual MS2 RNA strands over time scales from 1 ms to 900
s, we find that the start of assembly is broadly distributed in time and is
followed by a rapid increase in the number of bound proteins. These
measurements provide strong evidence for a nucleation-and-growth pathway. We
also find that malformed structures assemble when multiple nuclei appear on the
same RNA before the first nucleus has finished growing. Our measurements reveal
the complex assembly pathways for viral capsids around RNA in quantitative
detail, including the nucleation threshold, nucleation time, growth time, and
constraints on the critical nucleus size. These results may inform strategies
for engineering synthetic capsids or for derailing the assembly of pathogenic
viruses.
