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.