Measuring the selective packaging of RNA molecules by viral coat proteins in cells.

Some RNA viruses package their genomes with extraordinary selectivity, assembling protein capsids around their own viral RNA while excluding nearly all host RNA. How the assembling proteins distinguish viral RNA from host RNA is not fully understood, but RNA structure is thought to play a key role. To test this idea, we perform in-cellulo packaging experiments using bacteriophage MS2 coat proteins and a variety of RNA molecules in Significance Statement: Bacteriophage MS2 packages its RNA genome into protective protein shells called capsids while excluding nearly all host-cell RNA. Engineering synthetic capsids with similar selectivity could enable a broad range of RNA-based technologies, including CRISPR gene editing systems, mRNA vaccines, and other emerging RNA-based therapeutics. Our study shows that selective packaging in MS2 is not dictated by a single, high-affinity RNA-protein interaction but instead emerges from the collective interactions of multiple coat proteins and an ensemble of stem-loops distributed across the RNA molecule. By establishing these collective interactions as the basis of selectivity, our findings provide a foundation for engineering synthetic capsids capable of selectively packaging target RNAs for next-generation RNA-based technologies.