Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid.
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ABSTRACT:
SUBMITTER: Stockley PG
PROVIDER: S-EPMC4836477 | biostudies-other | 2016 Jan-Mar
REPOSITORIES: biostudies-other
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Project description:Using a combination of biochemistry, mass spectrometry, NMR spectroscopy and cryo-electron microscopy (cryo-EM), we have been able to show that quasi-equivalent conformer switching in the coat protein (CP) of an RNA bacteriophage (MS2) is controlled by a sequence-specific RNA-protein interaction. The RNA component of this complex is an RNA stem-loop encompassing just 19 nts from the phage genomic RNA, which is 3569 nts in length. This binding results in the conversion of a CP dimer from a symmetrical conformation to an asymmetric one. Only when both symmetrical and asymmetrical dimers are present in solution is assembly of the T = 3 phage capsid efficient. This implies that the conformers, we have characterized by NMR correspond to the two distinct quasi-equivalent conformers seen in the 3D structure of the virion. An icosahedrally-averaged single particle cryo-EM reconstruction of the wild-type phage (to, ~9 Å resolution) has revealed icosahedrally ordered density encompassing up to 90% of the single-stranded RNA genome. The RNA is seen with a novel arrangement of two concentric shells, with connections between them along the 5-fold symmetry axes. RNA in the outer shell interacts with each of the 90 CP dimers in the T = 3 capsid and although the density is icosahedrally averaged, there appears to be a different average contact at the different quasi-equivalent protein dimers: precisely the result that would be expected if protein conformer switching is RNA-mediated throughout the assembly pathway. This unprecedented RNA structure provides new constraints for models of viral assembly and we describe experiments aimed at probing these. Together, these results suggest that viral genomic RNA folding is an important factor in efficient assembly, and further suggest that RNAs that could sequester viral CPs but not fold appropriately could act as potent inhibitors of viral assembly.
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Project description:The all-atom model of an MS2 bacteriophage particle without its genome (the capsid) was built using high-resolution cryo-electron microscopy (EM) measurements for initial conformation. The structural characteristics of the capsid and the dynamics of the surrounding solution were examined using molecular dynamics simulation. The model demonstrates the overall preservation of the cryo-EM structure of the capsid at physiological conditions (room temperature and ions composition). The formation of a dense anion layer near the inner surface and a diffuse cation layer near the outer surface of the capsid was detected. The flow of water molecules and ions across the capsid through its pores were quantified, which was considerable for water and substantial for ions.
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Project description:Bacteriophage MS2 is a positive-sense, single-stranded RNA virus encapsulated in an asymmetric T = 3 pseudo-icosahedral capsid. It infects Escherichia coli through the F-pilus, in which it binds through a maturation protein incorporated into its capsid. Cryogenic electron microscopy has previously shown that its genome is highly ordered within virions, and that it regulates the assembly process of the capsid. In this study, we have assembled recombinant MS2 capsids with non-genomic RNA containing the capsid incorporation sequence, and investigated the structures formed, revealing that T = 3, T = 4 and mixed capsids between these two triangulation numbers are generated, and resolving structures of T = 3 and T = 4 capsids to 4 Å and 6 Å respectively. We conclude that the basic MS2 capsid can form a mix of T = 3 and T = 4 structures, supporting a role for the ordered genome in favouring the formation of functional T = 3 virions.
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Project description:Using cross-linking coupled to matrix-assisted laser desorption/ionization mass spectrometry and CLIP-Seq sequencing, we determined the peptide and oligonucleotide sequences at the interfaces between the capsid proteins and the genomic RNA of bacteriophage MS2. The results suggest that the same coat protein (CP)-RNA and maturation protein (MP)-RNA interfaces are used in every viral particle. The portions of the viral RNA in contact with CP subunits span the genome, consistent with a large number of discrete and similar contacts within each particle. Many of these sites match previous predictions of the locations of multiple, dispersed and degenerate RNA sites with cognate CP affinity termed packaging signals (PSs). Chemical RNA footprinting was used to compare the secondary structures of protein-free genomic fragments and the RNA in the virion. Some PSs are partially present in protein-free RNA but others would need to refold from their dominant solution conformations to form the contacts identified in the virion. The RNA-binding peptides within the MP map to two sections of the N-terminal half of the protein. Comparison of MP sequences from related phages suggests a similar arrangement of RNA-binding sites, although these N-terminal regions have only limited sequence conservation. In contrast, the sequences of the C-termini are highly conserved, consistent with them encompassing pilin-binding domains required for initial contact with host cells. These results provide independent and unambiguous support for the assembly of MS2 virions via a PS-mediated mechanism involving a series of induced-fit viral protein interactions with RNA.
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Project description:We have determined the structure to 2.8 A of an RNA aptamer (F5), containing 2'-deoxy-2-aminopurine (2AP) at the -10 position, complexed with MS2 coat protein by soaking the RNA into precrystallised MS2 capsids. The -10 position of the RNA is an important determinant of binding affinity for coat protein. Adenine at this position in other RNA stem-loops makes three hydrogen bonds to protein functional groups. Substituting 2AP for the -10 adenine in the F5 aptamer yields an RNA with the highest yet reported affinity for coat protein. The refined X-ray structure shows that the 2AP base makes an additional hydrogen bond to the protein compared to adenine that is presumably the principal origin of the increased affinity. There are also slight changes in phosphate backbone positions compared to unmodified F5 that probably also contribute to affinity. Such phosphate movements are common in structures of RNAs bound to the MS2 T = 3 protein shell and highlight problems for de novo design of RNA binding ligands.
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Project description:The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus-like particles. We applied our method to the coat proteins of bacteriophage MS2 as a specific model system. This virus-like particle of bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer, and the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than that between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles, and novel drug delivery systems.
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Project description:Experimental evolution of bacteriophage provides a powerful means of studying the genetics of adaptation, as every substitution contributing to adaptation can be identified and characterized. Here, I use experimental evolution of MS2, an RNA bacteriophage, to study its adaptive response to a novel environment. To this end, three lines of MS2 were adapted to rapid growth and lysis at cold temperature for a minimum of 50 phage generations and subjected to whole-genome sequencing. Using this system, I identified adaptive substitutions, monitored changes in frequency of adaptive mutations through the course of the experiment, and measured the effect on phage growth rate of each substitution. All three lines showed a substantial increase in fitness (a two- to threefold increase in growth rate) due to a modest number of substitutions (three to four). The data show some evidence that the substitutions occurring early in the experiment have larger beneficial effects than later ones, in accordance with the expected diminishing returns relationship between the fitness effects of a mutation and its order of substitution. Patterns of molecular evolution seen here--primarily a paucity of hitchhiking mutations--suggest an abundant supply of beneficial mutations in this system. Nevertheless, some beneficial mutations appear to have been lost, possibly due to accumulation of beneficial mutations on other genetic backgrounds, clonal interference, and negatively epistatic interactions with other beneficial mutations.
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Project description:Horizontal transfer of mobile genetic elements (MGEs) is a key aspect of the evolution of bacterial pathogens. Transduction by bacteriophages is especially important in this process. Bacteriophages-which assemble a machinery for efficient encapsidation and transfer of genetic material-often transfer MGEs and other chromosomal DNA in a more-or-less nonspecific low-frequency process known as generalized transduction. However, some MGEs have evolved highly specific mechanisms to take advantage of bacteriophages for their own propagation and high-frequency transfer while strongly interfering with phage production-"molecular piracy". These mechanisms include the ability to sense the presence of a phage entering lytic growth, specific recognition and packaging of MGE genomes into phage capsids, and the redirection of the phage assembly pathway to form capsids with a size more appropriate for the size of the MGE. This review focuses on the process of assembly redirection, which has evolved convergently in many different MGEs from across the bacterial universe. The diverse mechanisms that exist suggest that size redirection is an evolutionarily advantageous strategy for many MGEs.
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Project description:RNA is a polymer with pivotal functions in many biological processes. RNA structure determination is thus a vital step toward understanding its function. The secondary structure of RNA is stabilized by hydrogen bonds formed between nucleotide basepairs, and it defines the positions and shapes of functional stem-loops, internal loops, bulges, and other functional and structural elements. In this work, we present a methodology for studying large intact RNA biomolecules using homonuclear 15N solid-state NMR spectroscopy. We show that proton-driven spin-diffusion experiments with long mixing times, up to 16 s, improved by the incorporation of multiple rotor-synchronous 1H inversion pulses (termed radio-frequency dipolar recoupling pulses), reveal key hydrogen-bond contacts. In the full-length RNA isolated from MS2 phage, we observed strong and dominant contributions of guanine-cytosine Watson-Crick basepairs, and beyond these common interactions, we observe a significant contribution of the guanine-uracil wobble basepairs. Moreover, we can differentiate basepaired and non-basepaired nitrogen atoms. Using the improved technique facilitates characterization of hydrogen-bond types in intact large-scale RNA using solid-state NMR. It can be highly useful to guide secondary structure prediction techniques and possibly structure determination methods.
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Project description:We use all-atom simulations to elucidate the mechanisms underlying conformational switching and allostery within the coat protein of the bacteriophage MS2. Assembly of most icosahedral virus capsids requires that the capsid protein adopts different conformations at precise locations within the capsid. It has been shown that a 19 nucleotide stem loop (TR) from the MS2 genome acts as an allosteric effector, guiding conformational switching of the coat protein during capsid assembly. Since the principal conformational changes occur far from the TR binding site, it is important to understand the molecular mechanism underlying this allosteric communication. To this end, we use all-atom simulations with explicit water combined with a path sampling technique to sample the MS2 coat protein conformational transition, in the presence and absence of TR-binding. The calculations find that TR binding strongly alters the transition free energy profile, leading to a switch in the favored conformation. We discuss changes in molecular interactions responsible for this shift. We then identify networks of amino acids with correlated motions to reveal the mechanism by which effects of TR binding span the protein. We find that TR binding strongly affects residues located at the 5-fold and quasi-sixfold interfaces in the assembled capsid, suggesting a mechanism by which the TR binding could direct formation of the native capsid geometry. The analysis predicts amino acids whose substitution by mutagenesis could alter populations of the conformational substates or their transition rates.
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