The Three-dimensional Structure of Genomic RNA in Bacteriophage MS2: Implications for Assembly

Toropova, Katerina; Basnak, Gabriella; Twarock, Reidun; Stockley, Peter G.; Ranson, Neil A.

doi:10.1016/j.jmb.2007.08.067

Cited by 107 publications

(175 citation statements)

References 39 publications

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“…The complexation would not consist of continuous stretches of RNA, but rather would occur in the piecemeal fashion that we have just proposed for ss-RNA segments. 40,41 A similar mechanism has been proposed by Tihova et al,32 who suggested that base pairing between RNA segments in free solution is reversible and that there are a sufficient number of secondary structures of RNA from which to select one that is particularly wellsuited to formation of a dodecahedral cage.…”

Section: Resultsmentioning

confidence: 59%

Electrostatics of capsid-induced viral RNA organization

Forrey

Muthukumar

2009

The Journal of Chemical Physics

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We have addressed the role of electrostatics in the formation of genome structure in the Pariacoto virus, where substantial experimental data are available. We have used Langevin dynamics simulation of a coarse-grained model, based on the published crystal structure of the rigid portion of the Pariacoto capsid and including flexible N-terminal protein arms, attached to the rigid capsid at the appropriate locations. The inclusion of charged residues in our model was dictated solely by the location of charges inherent in the Pariacoto sequence itself. Although the viral genome and other exogenous RNA sequences used in experimental studies can assume secondary structures, we have intentionally used uniformly charged flexible polyelectrolyte lacking predetermined secondary structures as the substitute for the viral genome, in order to see whether the same final assembled genome structure emerges without invoking secondary RNA structures. The intent of our study was to investigate the internal environment presented by the capsid proteins of Pariacoto virus, specifically whether the topological features and electrostatic potential at the inner capsid surface can induce complexation of generic negatively charged polyelectrolyte into structures similar to those observed experimentally with packaged RNA. We find that the charge decoration on the interior of the capsid templates the assembly of the flexible polyelectrolyte, allowing hybridizationlike folding of similarly charged strands, and eventually organizing dodecahedral assembly of the polymer. Our results from a generic flexible polyelectrolyte for the assembled structure and bimodal monomer distribution are remarkably matched to that of the viral RNA found experimentally. Results of our work can be interpreted primarily as a consequence of electrostatics, as consideration of base-pairing has been omitted. We propose that our work supports the growing body of evidence that electrostatic interactions play a crucial role in RNA viral assembly and structure.

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Section: Resultsmentioning

confidence: 59%

Electrostatics of capsid-induced viral RNA organization

Forrey

Muthukumar

2009

The Journal of Chemical Physics

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“…The high-resolution X-ray structure of the MS2 virion, like many ssRNA viruses, does not show any density that can be ascribed to the viral RNA (Valegå rd et al, 1990). However, an icosahedrally averaged cryo-EM structure at intermediate ($9 Å ) resolution reveals a double shell of RNA packaged with approximate order (Toropova et al, 2008). Strikingly, once again, array points overlapping with the volume occupied by the RNA map around the contours of this RNA density (Figs.…”

Section: Genome Organization Is Also Predictablementioning

confidence: 92%

“…This is even more striking given that yellow and orange points are predicted to be located at different radial levels, corresponding to the contacts with the two different types of dimeric building blocks (A/B and C/C) of the capsid. (c) An illustration of the match with the cryo-EM RNA density (Toropova et al, 2008), shown here as a radially coloured transparent surface. Array points map the inside (maroon points, radius of 38 Å ) and outside (blue and mid-blue points, 62 and 65 Å , respectively) surfaces of the inner RNA shell, and also mark the density connecting the inner and outer RNA shells (cyan, 75 Å ).…”

Section: Application To a Non-quasi-equivalent Virusmentioning

confidence: 99%

“…Striking examples of this are the dodecahedral cage of dsRNA seen in Pariacoto virus (PaV) (Tang et al, 2001) and the double-shell architecture of the viral RNA observed in bacteriophage MS2 (Toropova et al, 2008). These results suggest that there are further constraints on the threedimensional structures of these highly ordered capsids and genomes, i.e.…”

Section: Introductionmentioning

confidence: 99%

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Structural constraints on the three-dimensional geometry of simple viruses: case studies of a new predictive tool

Keef

Wardman

Ranson

et al. 2013

Acta Crystallogr A Found Crystallogr

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Understanding the fundamental principles of virus architecture is one of the most important challenges in biology and medicine. Crick and Watson were the first to propose that viruses exhibit symmetry in the organization of their protein containers for reasons of genetic economy. Based on this, Caspar and Klug introduced quasi-equivalence theory to predict the relative locations of the coat proteins within these containers and classified virus structure in terms of T-numbers. Here it is shown that quasi-equivalence is part of a wider set of structural constraints on virus structure. These constraints can be formulated using an extension of the underlying symmetry group and this is demonstrated with a number of case studies. This new concept in virus biology provides for the first time predictive information on the structural constraints on coat protein and genome topography, and reveals a previously unrecognized structural interdependence of the shapes and sizes of different viral components. It opens up the possibility of distinguishing the structures of different viruses with the same T-number, suggesting a refined viral structure classification scheme. It can moreover be used as a basis for models of virus function, e.g. to characterize the start and end configurations of a structural transition important for infection.

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“…The first glimpse of RNA within the capsid came from a low-resolution structure of MS2 using single-particle cryo-EM (27). On the basis of another single-particle cryo-EM structure with icosahedral symmetry applied, it was proposed that MS2 has two concentric shells of RNA within the phage capsid (28). Subsequent work, using cryo-electron tomography and subtomogram averaging, yielded a 39-Å resolution symmetry-free density map of MS2 (29), suggesting the gRNA adopts the same conformation for each virus particle within the capsid and the maturation protein replaces a C/C dimer of the coat proteins.…”

mentioning

confidence: 99%

Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

Gorzelnik

Zhao

Reed

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Single-stranded (ss) RNA viruses infect all domains of life. To date, for most ssRNA virions, only the structures of the capsids and their associated protein components have been resolved to high resolution. Qβ, an ssRNA phage specific for the conjugative F-pilus, has a T = 3 icosahedral lattice of coat proteins assembled around its 4,217 nucleotides of genomic RNA (gRNA). In the mature virion, the maturation protein, A 2 , binds to the gRNA and is required for adsorption to the F-pilus. Here, we report the cryo-electron microscopy (cryo-EM) structures of Qβ with and without symmetry applied. The icosahedral structure, at 3.7-Å resolution, resolves loops not previously seen in the published X-ray structure, whereas the asymmetric structure, at 7-Å resolution, reveals A 2 and the gRNA. A 2 contains a bundle of α-helices and replaces one dimer of coat proteins at a twofold axis. The helix bundle binds gRNA, causing denser packing of RNA in its proximity, which asymmetrically expands the surrounding coat protein shell to potentially facilitate RNA release during infection. We observe a fixed pattern of gRNA organization among all viral particles, with the major and minor grooves of RNA helices clearly visible. A single layer of RNA directly contacts every copy of the coat protein, with one-third of the interactions occurring at operator-like RNA hairpins. These RNA-coat interactions stabilize the tertiary structure of gRNA within the virion, which could further provide a roadmap for capsid assembly.single-particle cryo-EM | Allolevivirus | ssRNA virus | genomic RNA | maturation protein S ingle-stranded (ss) RNA viruses are an abundant type of virus and infect all domains of life (1-4). One of the beststudied ssRNA virus systems is the Leviviridae, which infects Gram-negative bacteria via a variety of retractile pili (5); extensive genetic and biochemical studies have been performed on two of these phages: MS2 and Qβ (5-11). All of the Leviviridae have the same core genome, spanning 3.4-4.3 kb, encoding the maturation protein, the coat protein, and a subunit of RNAdependent RNA replicase (SI Appendix, Fig. S1) (10). The MS2-like phages, designated true leviviruses, have a fourth gene that encodes the lysis protein, whereas the Qβ-like phages, designated alloleviviruses, have the lysis function as an additional feature of the maturation protein (called A 2 in Qβ). Qβ also encodes a minor coat protein, called A 1 , arising from occasional readthrough of the stop codon of the major coat protein; it has been estimated that the A 1 protein replaces 3-10 copies of the major coat protein in the virion (11) and is required for infection (12). A 1 consists of a coat domain and a read-through domain separated by a flexible linker (13). Unlike most dsDNA phages, which use specialized protein machinery to pump their genomic DNA into a capsid preassembled around a protein scaffold (14-17), ssRNA viruses, including the Leviviridae, assemble their coat proteins around the genomic RNA (gRNA), presumably because the extremely small ...

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The Three-dimensional Structure of Genomic RNA in Bacteriophage MS2: Implications for Assembly

Cited by 107 publications

References 39 publications

Electrostatics of capsid-induced viral RNA organization

Electrostatics of capsid-induced viral RNA organization

Structural constraints on the three-dimensional geometry of simple viruses: case studies of a new predictive tool

Asymmetric cryo-EM structure of the canonical Allolevivirus Qβ reveals a single maturation protein and the genomic ssRNA in situ

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