Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20–30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA–protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein–protein interactions, consistent with the RNA–protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA–coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.

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Long RNA molecules are at the core of gene regulation across all kingdoms of life, while also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with nonrepeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. To test whether a branched polymer model can estimate the overall sizes of large RNAs, we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long noncoding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation-one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of maximum ladder distance. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low ionic-strength sizes. These results suggest that sizes of long RNA molecules are determined by the branching pattern of their secondary structures. We also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs.

Viroplasms are cytoplasmic, membraneless structures assembled in rotavirus (RV)-infected cells, which are intricately involved in viral replication. Two virus-encoded, non-structural proteins, NSP2 and NSP5, are the main drivers of viroplasm formation. The structures (as far as is known) and functions of these proteins are described. Recent studies using plasmid-only-based reverse genetics have significantly contributed to elucidation of the crucial roles of these proteins in RV replication. Thus, it has been recognized that viroplasms resemble liquid-like protein–RNA condensates that may be formed via liquid–liquid phase separation (LLPS) of NSP2 and NSP5 at the early stages of infection. Interactions between the RNA chaperone NSP2 and the multivalent, intrinsically disordered protein NSP5 result in their condensation (protein droplet formation), which plays a central role in viroplasm assembly. These droplets may provide a unique molecular environment for the establishment of inter-molecular contacts between the RV (+)ssRNA transcripts, followed by their assortment and equimolar packaging. Future efforts to improve our understanding of RV replication and genome assortment in viroplasms should focus on their complex molecular composition, which changes dynamically throughout the RV replication cycle, to support distinct stages of virion assembly.

Segmented RNA viruses are ubiquitous pathogens, which include influenza viruses and rotaviruses. A major challenge in understanding their assembly is the combinatorial problem of a non-random selection of a full genomic set of distinct RNAs. This process involves complex RNA-RNA and protein-RNA interactions, which are often obscured by non-specific binding at concentrations approaching in vivo assembly conditions. Here, we present direct experimental evidence of sequence-specific inter-segment interactions between rotavirus RNAs, taking place in a complex RNA- and protein-rich milieu. We show that binding of the rotavirus-encoded non-structural protein NSP2 to viral ssRNAs results in the remodeling of RNA, which is conducive to formation of stable inter-segment contacts. To identify the sites of these interactions, we have developed an RNA-RNA SELEX approach for mapping the sequences involved in inter-segment base-pairing. Our findings elucidate the molecular basis underlying inter-segment interactions in rotaviruses, paving the way for delineating similar RNA-RNA interactions that govern assembly of other segmented RNA viruses.