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

Abstract

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.

Physics of viral dynamics

“… 2 ) provided us with a systematic classification of icosahedral capsids in terms of the so-called T -numbers ( triangulation numbers ), a method that has stood the test of time 3 . More recently, modern cryo-electron imaging methods, including tomography 4 , 5 and asymmetric reconstructions 6 , 7 , have made it possible to reconstruct not only regular capsids, but also asymmetric protein distributions and the enclosed genome by using images of individual viruses.…”

Perspectives on Viral RNA Genomes and the RNA Folding Problem

“…In the case of icosahedral virus particles, the outside surface of the virus particle is symmetric and prevents the experimental purifying of different internal conformations of the viral genome. Two examples of high-resolution asymmetric reconstructions of MS2 bacteriophage [ 4 ] and Brome Mosaic Virus (BMV) RNA genomes [ 5 ] highlight two different models for viral genome structure and virus assembly mechanisms.…”

“…Most recently, the high-resolution asymmetric reconstruction of BMV shows a partially ordered ensemble of conformations for the viral RNA genome [ 5 ]. BMV has 4 distinct RNAs that compose its genome.…”

“…Since the first observations of encapsidated RNA genomes, there have been many approaches to determining and modelling the structures of viral RNA genomes. This review will focus on two new techniques, asymmetric reconstructions from high resolution cryoelectron microscopy [ 4 , 5 ] and DREEM (Detection of RNA folding Ensembles using Expectation Maximization) [ 6 ], that probe the structures of encapsidated RNA. These techniques provide positive evidence that encapsidated RNA forms more than one structure inside a virus particle.…”

“…Different models for Satellite Tobacco Mosaic Virus RNA have proposed an ensemble of structures [ 12 ] or a single minimum energy model [ 13 , 14 ]. Several models for MS2 bacteriophage RNA have proposed an ensemble of structures [ 15 ], models with regions of order and regions of disorder [ 4 , 5 ], or single minimum free energy structures [ 16 ].…”