We report the asymmetric reconstruction of the single-stranded RNA (ssRNA) content in one of the three otherwise identical virions of a multipartite RNA virus, brome mosaic virus (BMV). We exploit a sample consisting exclusively of particles with the same RNA content—specifically, RNAs 3 and 4—assembled in planta by agrobacterium-mediated transient expression. We find that the interior of the particle is nearly empty, with most of the RNA genome situated at the capsid shell. However, this density is disordered in the sense that the RNA is not associated with any particular structure but rather, with an ensemble of secondary/tertiary structures that interact with the capsid protein. Our results illustrate a fundamental difference between the ssRNA organization in the multipartite BMV viral capsid and the monopartite bacteriophages MS2 and Qβ for which a dominant RNA conformation is found inside the assembled viral capsids, with RNA density conserved even at the center of the particle. This can be understood in the context of the differing demands on their respective lifecycles: BMV must package separately each of several different RNA molecules and has been shown to replicate and package them in isolated, membrane-bound, cytoplasmic complexes, whereas the bacteriophages exploit sequence-specific “packaging signals” throughout the viral RNA to package their monopartite genomes.

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Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids. IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

The family Bromoviridae contains six genera of tripartite, plant‐pathogenic ribonucleic acid (RNA) viruses, namely Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus and Oleavirus . Viruses in this family exhibit the following salient features: (1) virions are nonenveloped with icosahedral or bacilliform geometries; (2) in each genus, the single‐strand, positive‐sense RNA genome of ∼8 kilobases (kb) is divided among three RNAs; (3) the 3' untranslated region of the viral RNAs adopts either a transfer ribonucleic acid‐like structure (TLS) or a series of stem loops that assume a TLS upon coat protein (CP) binding; (4) four progeny RNAs are packaged into either three homogeneous or four heterogeneous virions; (5) CP is required for the genome activation exclusively for Alfamovirus and Ilarvirus genera; (6) in all the genera, the replication and virion assembly occurs in the cytoplasmic compartment of the cell and (7) cell‐to‐cell and long‐distance spread involves both movement protein and CP. Finally, members of Bromoviridae and other plant viruses commonly share mechanical and insect transmission properties. Key Concepts The genome of the member viruses of the family Bromoviridae contains single‐strand, positive‐sense RNAs encoding few proteins. A group of three RNA components represents the genome in tripartite viruses. Vesicles or spherule‐like invaginations of host membranes induced by virus‐encoded gene products are the sites of viral replication. Progeny RNA of tripartite viruses is packaged into three or four virions having either spherical or bacilliform morphology. Genome packaging into stable virions requires specific interaction between the coat protein (CP) and the viral progeny RNA and is functionally coupled to replication, that is only the replicated progeny RNA is packaged into virions. Cell‐to‐cell spread of the tripartite viruses requires both movement protein and CP.

Infectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. In viruses, conformational changes are essential for establishing infection and evolution.