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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 inEscherichia coli. In each experiment, plasmid-derived RNA molecules with a specified sequence compete against the cellular transcriptome for packaging by plasmid-derived coat proteins. Following this competition, we quantify the total amount and relative composition of the packaged RNA using electron microscopy, interferometric scattering microscopy, and high-throughput sequencing. By systematically varying the input RNA sequence and measuring changes in packaging outcomes, we are able to directly test competing models of selective packaging. Our results rule out a longstanding model in which selective packaging requires the well-known translational repressor (TR) stem-loop, and instead support more recent models in which selectivity emerges from the collective interactions of multiple coat proteins and multiple stem-loops distributed across the RNA molecule. These findings establish a framework for studying and understanding selective packaging in a range of natural viruses and virus-like particles, and lay the groundwork for engineering synthetic systems that package specific RNA cargoes. 

Abstract We describe a DNA-array-based method to infer intramolecular connections in a population of RNA molecules in vitro. First we add DNA oligonucleotide “patches” that perturb the RNA connections, and then we use a microarray containing a complete set of DNA oligonucleotide “probes” to record where perturbations occur. The pattern of perturbations reveals couplings between regions of the RNA sequence, from which we infer connections as well as their prevalences in the population, without reference to folding models. We validate this patch–probe method using the 1058-nucleotide RNA genome of satellite tobacco mosaic virus (STMV), which has been shown to have multiple long-range connections. Our results not only indicate long-range connections that agree with previous structures but also reveal the prevalence of competing connections. Together, these results suggest that multiple structures with different connectivity coexist in solution. Furthermore, we show that the prevalence of certain connections changes when pseudouridine, an important component of natural and synthetic RNAs, is substituted for uridine in STMV RNA, and that the connectivity of STMV minus strands is qualitatively distinct from that of plus strands. Finally, we use a simplified version of the method to validate a predicted 317-nucleotide connection within the 3569-nucleotide RNA genome of bacteriophage MS2. 

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 more than 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, but the time to grow a capsid after nucleation is largely unaffected. These results suggest 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 intraprotein 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.