Modulation of Capsid Dynamics in Bromoviruses by the Host and Heterologous Viral Replicase

The tripartite genome of Cowpea chlorotic mottle virus is packaged into three morphologically indistinguishable icosahedral virions with T=3 symmetry. The two virion types, C1V and C2V, package genomic RNAs 1 (C1) and 2 (C2), respectively. The third virion type, C3+4V, co-packages genomic RNA3 and its sub-genomic RNA (RNA4). In this study, each virion type was separately assembled in planta using a previously established agroinfiltration approach. The three virion types are indistinguishable under the EM and by electrophoretic mobility. However, the stability and the capsid dynamics evaluated respectively by the differential scanning fluorimetry and MALDI-TOF analysis following trypsin digestion distinguished the three virions type. Proteolytic analysis revealed that, while retaining the structural integrity, C1V and C2V virions released peptide regions encompassing the N-terminal arginine-rich RNA binding motif. In contrast, a minor population of the C3+4V virion type was sensitive to trypsin-releasing peptides encompassing the entire capsid protein region. In addition, we also evaluated the effect of the two host organisms on the capsid dynamics. The wild-type CCMV virions purified from cowpea are highly susceptible to trypsin digestion, while those from N. benthamiana remained resistant. Finally, the MALDI-TOF evaluated the relative dynamics of C3+4V and B3+4V virions assembled under the control of the homologous vs. heterologous replicase. The significance of the viral replicase in modulating the capsid dynamics was evident by the differential sensitivity to protease exhibited by B3+4V and C3+4V virions assembled under the homologous vs. heterologous replicase. The significance of these results in relation to viral biology is discussed. IMPORTANCEInfectious virus particles or virions are considered static structures and undergo various conformational transitions to replicate and infect many eukaryotic cells. Although viral capsid fluctuations, referred to as dynamics or breathing, have been well studied in RNA viruses pathogenic to animals, such information is limited among plant viruses. Cowpea chlorotic mottle virus (CCMV) and Brome mosaic virus (BMV), RNA viruses pathogenic to plants, contain a segmented genome distributed among three physically and structurally homogenous icosahedral virion types. This study examines the role of the host organism and the heterologous viral replicase in modulating the thermal stability and capsid dynamics in CCMV and BMV. The results accentuate that alteration of the capsid dynamics by the host type and heterologous replicase are likely to affect the biology of the virus.