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

Abstract

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.

Subset of Fluorophores Is Responsible for Radiation Brightening in Viromimetic Particles

“…This was followed by limited proteolysis. , MALDI-TOF peptide mapping helped pinpointing the cleavage site. A similar characterization method was previously employed in studying the coat protein N-terminus and C terminus dynamics of other icosahedral viruses. ,, …”

“…Amide hydrogen exchange studies suggest that the BMV capsid is characterized by rich dynamics. , The relative solvent accessibility of BMV lysines was explored by means of chemical labeling and tandem mass spectrometry . Such bottom-up proteomics approach involves enzymatic digestion combined with peptide mapping and has proven successful for rapid virus identification and elucidation of fluorophore modification sites in proteins. , Here, we probe the fluorophore conjugation sites on the BMV capsid by employing a similar proteomics method to elucidate the active distribution of the fluorescein derivative commercially known as Oregon Green (OG).…”

Controlling the surface charge of simple viruses

“…For CCMV, the virus and cleaved protein pIs are also close to one another (3.7 and 4.1). Similarly, we find that the gel EMs of wt and empty BMV capsids are the same: see Fig 3 . Johnson, et al [13] had also found that the solution EMs of CCMV and BMV virions are controlled by their surface charge and more recently, Chakravarty, et al [26] have shown that BMV virions engineered to contain either BMV RNA 1 (3200 nt), 2 (2800 nt) or 3+4 (2900 nt) have indistinguishable gel EMs at pH 4 and are negatively charged.…”

Genome organization and interaction with capsid protein in a multipartite RNA virus

“…X-ray crystallography of CCMV has shown that residues 34,42,45,47,48,82,85,87,89,140,143,179,181, and 184 interact with density that was attributed to RNA (7), while recent studies using Clip-Seq analysis (13,39)-in which the binding footprints of CP for RNA and vice versa are investigated by cross-linking capsids with different RNA cargo-have found that over 60% of the CP interacts with RNA. We find that residues 34, 42 to 48, 82 to 89, and 140 to 143 in BMV (Figs.…”

“…We are unable to ascertain to what extent the packaging of two molecules (RNAs 3 and 4) rather than a single one (RNA1 or RNA2) affects the extent to which RNA is ordered in the capsid. Recent experiments involving trypsin-protease digestion of B1, B2, and B3 + 4 virions analyzed by both western blot analysis and MALDI-TOF show that B1 and B2 virions exhibit a distinct protease digestion profile from B3 + 4 virions, suggesting that the packaged RNA affects capsid dynamics (47). Accordingly, we plan to carry out high-resolution reconstructions, similar to those reported here, with in planta-prepared BMV1 and BMV2 virions to determine differences in RNA ordering when a single RNA molecule is inside.…”