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Bacteriophage MS2 Genomic RNA Encodes an Assembly Instruction Manual for Its Capsid This is a repository copy of Bacteriophage MS2 Genomic RNA Encodes an Assembly Instruction Manual for Its Capsid. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/99359/ Version: Accepted Version Article: Stockley, Peter G., White, Simon J., Dykeman, Eric Charles et al. (11 more authors) (2016) Bacteriophage MS2 Genomic RNA Encodes an Assembly Instruction Manual for Its Capsid. Bacteriophage. e1157666. ISSN 2159-7081 https://doi.org/10.1080/21597081.2016.1157666 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Bacteriophage ISSN: (Print) 2159-7081 (Online) Journal homepage: http://www.tandfonline.com/loi/kbac20 Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid Peter G. Stockley, Simon J. White, Eric Dykeman, Iain Manfield, Ottar Rolfsson, Nikesh Patel, Richard Bingham, Amy Barker, Emma Wroblewski, Rebecca Chandler-Bostock, Eva U. Weiß, Neil A. Ranson, Roman Tuma & Reidun Twarock To cite this article: Peter G. Stockley, Simon J. White, Eric Dykeman, Iain Manfield, Ottar Rolfsson, Nikesh Patel, Richard Bingham, Amy Barker, Emma Wroblewski, Rebecca Chandler- Bostock, Eva U. Weiß, Neil A. Ranson, Roman Tuma & Reidun Twarock (2016) Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid, Bacteriophage, 6:1, e1157666, DOI: 10.1080/21597081.2016.1157666 To link to this article: http://dx.doi.org/10.1080/21597081.2016.1157666 © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC© Peter G. Stockley, Simon J. White, Eric Dykeman, Iain Manfield, Ottar Rolfsson, AcceptedNikesh Patel, author Richard version Bingham, posted Amy online: Barker, 02 MarEmma 2016. Wroblewski, Rebecca Chandler- Bostock, Eva U. Weiß, Neil A. Ranson, Roman Tuma, and Reidun Twarock Submit your article to this journal Article views: 81 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kbac20 Download by: [University of York] Date: 05 May 2016, At: 09:21 BACTERIOPHAGE 2016, VOL. 6, NO. 1, e1157666 (8 pages) http://dx.doi.org/10.1080/21597081.2016.1157666 ADDENDUM Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid Peter G. Stockleya, Simon J. Whitea, Eric Dykemanb, Iain Manfielda, Ottar Rolfssonc, Nikesh Patela, Richard Binghamb, Amy Barkera, Emma Wroblewskia, Rebecca Chandler-Bostocka, Eva U. Weißb, Neil A. Ransona, Roman Tumaa, and Reidun Twarockb aAstbury Center for Structural Molecular Biology, University of Leeds, Leeds, UK; bDepartment of Biology and Mathematics & York Center for Complex Systems Analysis, University of York, York, UK; cCenter for Systems Biology, University of Iceland, University of Iceland Biomedical Center, Reykjavik, Iceland ABSTRACT ARTICLE HISTORY Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on Received 12 January 2016 the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity Revised 15 February 2016 translational operator, TR, within the phage genome. The data also reveal the sequences of Accepted 17 February 2016 genomic fragments in contact with the coat protein shell. These show remarkable overlap with KEYWORDS previous predictions based on the hypothesis that virion assembly is mediated by multiple RNA bacteriophage; RNA sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on packaging signals; virion the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the assembly dominant assembly pathway and ensure cognate RNA encapsidation. In MS2, they also trigger conformational change in the dimeric capsomere creating the A/B quasi-conformer, 60 of which are needed to complete the TD3 capsid. This is the most compelling demonstration to date that this ssRNA virus, and by implications potentially very many of them, assemble via a PS-mediated assembly mechanism. Background (TBSV), was revealed by Harrison and co-workers in a Single-stranded, positive-sense RNA phages co-assem- technical tour de force they defined the major struc- ble their protective coat protein shells around copies of tural questions that needed to be answered to explain their genomic RNA. This is one of the 2 common virus that structure.8 These are how is genome encapsidation assembly mechanisms, the other being formation of a specificity achieved, and secondly how is the quasi- procapsid and the subsequent introduction of the viral equivalence of the TBSV TD3 capsid established dur- genome, most commonly in the form of dsDNA, via ing assembly. We have now provided mechanistic 9-12 Downloaded by [University of York] at 09:21 05 May 2016 specialized protein machinery. Spontaneous co-assem- explanations for both these events. We have shown bly is an essential feature of the lifecycles of a very that multiple RNA sites encompassing minimal CP wide range of important pathogens in every kingdom sequence recognition motifs, when presented via RNA of life.1 Genome packaging in these viruses has been folding, regulate both packaging specificity and the thought to be dominated by coat protein-coat protein pathway of virion assembly (Fig. 1). In effect, the RNA (CP) interactions, and favorable electrostatic contacts encodes a set of capsid assembly instructions, via mul- that ensure RNA encapsidation, with at most a single, tiple Packaging Signals (PSs). PSs facilitate formation high-affinity CP-binding RNA signal.2-6 Recent work of the protein-protein contacts in their capsids, and for with a variety of viruses in this family suggests that bacteriophage MS2 it has been shown that they also these models are not representative of in vivo scenar- act as allosteric switches determining the formation ios.7 When the first atomic structure of a spherical and placement of the quasi-conformers of the capso- virus, namely that of Tomato Bushy Stunt Virus meres forming the TD3shell13-17 (Fig. 2). CONTACT Peter G. Stockley [email protected] Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK. Addendum to: Direct Evidence for Packaging Signal-mediated Assembly of Bacteriophage MS2. O. Rolfsson, S. Middleton, I. W. Manfield, S. J. White, B. Fan, R. Vaughan, N. A. Ranson, E. Dykeman, R. Twarock, J. Ford, C. C. Kao & P. G. Stockley. J Mol Biol, 2015: doi: 10.1016/j.jmb.2015.11.014. © 2016 Peter G. Stockley, Simon J. White, Eric Dykeman, Iain Manfield, Ottar Rolfsson, Nikesh Patel, Richard Bingham, Amy Barker, Emma Wroblewski, Rebecca Chandler-Bostock, Eva U. Weiß, Neil A. Ranson, Roman Tuma, and Reidun Twarock. Published with license by Taylor & Francis. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. e1157666-2 P. G. STOCKLEY ET AL. Downloaded by [University of York] at 09:21 05 May 2016 Figure 1. PS-mediated assembly of STNV. (A) Multiple, short, degenerate CP-recognition motifs within the STNV genome (highlighted in red) in the primary sequence (top), when presented appropriately by RNA folding into stem-loops, are bound co-operatively at high affinity (low nM) by cognate CPs. This is revealed by smFCS assays with oligos dye-labeled at one end (middle). An individual PS, shown to the left of the smFCS trace in blue, stimulates sequence-specific assembly when titrated with increasing CP concentrations. Titration points are shown below the trace also in blue. At a threshold concentration, »5 nM, the Rh shifts from around 3 nm to around 7 nm, consistent with formation of an RNA-CP capsomere, which we have shown contains 3 CP subunits. Thereafter, a T D 1 VLP is created, due to CP-CP interactions with this initiation complex that is only complete by 3 mM CP. In contrast, a viral fragment encompassing the first 127 nt at the 50 end of the genome and predicted to form 5 PSs, reveals the co-operativity between these CP binding sites. The ini- tial Rh is unchanged until the CP concentration reaches »120 nM, where it declines by about 20% mimicking effects we have seen on the full length genome (titration points shown in black above the trace). As CP concentration increases there is a rapid and complete transition to a T D 1 capsid, reflecting the co-operative interactions between CPs being mediated by the PSs. There are at least 2 stages of assembly, a rapid initial collapse of the genome preparing it for encapsidation into the limited space of its TD1 capsid (cartoon, bot- tom), and a slower assembly completion stage. (B) The molecular basis of PS action is revealed by the crystal structure of VLPs assem- bled around an RNA encompassing a high affinity PS, B3. In the presence of PSs, a region of the CP toward the N-terminus that is normally disordered forms an additional turn of a-helix. This region is rich in basic amino acids suggesting that PS binding overcomes an electrostatic barrier preventing CP-CP interaction from forming the trimeric capsomere. BACTERIOPHAGE e1157666-3 Figure 2. PS-mediated assembly of the T D 3 MS2 virion. (A) The capsomere in this case is a CP dimer, which in the absence of RNA is symmetrical in solution, consistent with it resembling the C/C quasi-dimer of the capsid.
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