Single-particle studies of the effects of RNA-protein interactions on the self-assembly of RNA virus particles

Abstract

Understanding the pathways by which simple RNA viruses self-assemble from their coat proteins and RNA is of practical and fundamental interest. Although RNA-protein interactions are thought to play a critical role in the assembly, our understanding of their effects is limited because the assembly process is difficult to observe directly. We address this problem by using interferometric scattering microscopy, a sensitive optical technique with high dynamic range, to follow the in vitro assembly kinetics of over 500 individual particles of brome mosaic virus (BMV)--for which RNA-protein interactions can be controlled by varying the ionic strength of the buffer. We find that when RNA-protein interactions are weak, BMV assembles by a nucleation-and-growth pathway in which a small cluster of RNA-bound proteins must exceed a critical size before additional proteins can bind. As the strength of RNA-protein interactions increases, the nucleation time becomes shorter and more narrowly distributed until the assembly kinetics become indistinguishable from diffusion-limited adsorption. In contrast, the time to grow a capsid after nucleation varies weakly with both salt and protein concentration. These results show that the nucleation rate is controlled by RNA-protein interactions, while the growth process is driven less by RNA-protein interactions and more by protein-protein interactions and intra-protein forces. The nucleated pathway observed with the plant virus BMV is strikingly similar to that previously observed with bacteriophage MS2, a phylogenetically distinct virus with a different host kingdom. These results raise the possibility that nucleated assembly pathways might be common to other RNA viruses. RNA viruses first inspired the term "self-assembly." Yet much is still not understood about how even the simplest such viruses assemble or if different viruses assemble in similar ways. Theoretical models suggest many possible assembly pathways, with many different roles for the RNA, but until recently measuring these pathways has not been possible. We use a sensitive microscopy technique to follow the assembly of individual particles of BMV, a plant virus. We find evidence of an RNA-mediated nucleation-and-growth pathway that is strikingly similar to that of MS2, a bacterial virus. The last common ancestor of BMV and MS2 existed only in ancient times, suggesting that their assembly pathway might be evolutionarily conserved and other viruses might follow a similar pathway.