Rao, A. L. N. | Search bioRxiv

bioRxiv2026

Subgenomic RNA Co-packaging Links Capsid Dynamics to Cell-to-Cell Movement in Brome Mosaic Virus

Subgenomic RNAs (sgRNAs) regulate gene expression in many positive-strand RNA viruses, yet their functional contributions beyond protein coding remain poorly understood. Here, we investigate how incorporation of subgenomic RNA4 (sgRNA4) into Brome mosaic virus (BMV) virions is associated with changes in virion structure, stability, and viral movement. Using an in vivo assembly system, we generated two distinct virion populations: particles containing RNA3 and sgRNA4 (B3+4V) and particles containing RNA3 alone (B3V). Although the two virion types were physically indistinguishable by electron microscopy, they exhibited strikingly different biochemical and biological properties. B3+4V virions displayed increased protease sensitivity and structural flexibility, whereas B3V virions were protease resistant and structurally rigid. MALDI-time of flight analysis revealed that trypsin digestion released similar peptide fragments from both virion types, while a persistent intact capsid protein peak in B3V preparations indicated that only a small fraction of particles was accessible to proteolysis. In functional assays in Nicotiana benthamiana, B3+4V virions were efficiently detected in adjacent tissues beyond the primary infiltration sites, whereas B3V virions showed little to no cell-to-cell spread beyond the primary infiltration sites. Together, these results support a model in which sgRNA4 co-packaging is functionally linked to capsid dynamics that correlate with efficient viral movement. More broadly, our findings indicate that differences in packaged RNA composition can modulate virion physical properties and biological behavior, providing insight into how RNA viruses coordinate genome organization, assembly, and intercellular spread. ImportanceThis study reveals that packaging of the small subgenomic RNA (sgRNA4) within BMV particles plays a crucial role beyond assembly--it is strongly associated with how the virus spreads between plant cells. Virions containing sgRNA4 are more flexible and dynamic, correlating with efficient movement through cell connections, while those lacking it are rigid and movement-deficient. These results uncover an unexpected link between RNA content and viral mobility, showing that genome composition can fine-tune both structural and biological properties of viruses--a principle that may extend to many other RNA viruses affecting plants and animals.

microbiology↗

bioRxiv2019

Unravelling the Stability and Capsid Dynamics of the Three Virions of Brome Mosaic Virus Assembled Autonomously In Vivo

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replicated three-molecule RNA progeny of Brome mosaic virus (BMV) are packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasi-symmetry. Type 1 (B1v) and type 2 (B2v) virions respectively package genomic RNA1 or RNA2, while type 3 (B3+4v) co-packages genomic RNA3 (B3) and its sub-genomic RNA4 (B4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta. Physical and biochemical approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types, so protease-based mapping experiments were used to analyze the conformational dynamics of the individual virions. The crystallographic structure of the BMV capsid shows four trypsin-cleavage sites (K65, R103, K111 and K165 on the A, B and C subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1v and B2v released only two peptides involving amino acids 2-8 and 16-22 from the N-proximal arginine-rich RNA binding motif. In contrast, B3+4v capsids are unstable to trypsin, releasing several peptides in addition to the four sites predicted to be 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. IMPORTANCEThe 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 (RNAs 1, 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 capsid dynamics by 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 RNAs1 and 2 are more stable and dynamically distinct from those co-packaging RNA3 and 4, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.

molecular biology↗