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Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid.

Stockley PG, White SJ, Dykeman E, Manfield I, Rolfsson O, Patel N, Bingham R, Barker A, Wroblewski E, Chandler-Bostock R, Weiß EU, Ranson NA, Tuma R, Twarock R - Bacteriophage (2016)

Bottom Line: They act co-operatively to regulate the 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 T=3 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.

View Article: PubMed Central - PubMed

Affiliation: Astbury Center for Structural Molecular Biology, University of Leeds , Leeds, UK.

ABSTRACT

Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the 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 T=3 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.

No MeSH data available.


Related in: MedlinePlus

PS-mediated assembly of the T = 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. Binding to TR, and other related stem-loops, triggers a conformational change to the A/B-like dimer. Such RNA switching is required up to 60 times to make the T = 3 shell. (B) There is only a single copy of TR within the MS2 genome but many variant stem-loops can be formed that contain some of the important recognition features for CP31. Note, when these interactions (arrowed in figure) are measured in vitro their affinities are lowered significantly (shown as relative reduced affinities compared to TR). However, the concentration of a single stem-loop in the volume of the capsid is ˜0.3 mM. This compares with the Kd for TR binding to a CP dimer which is 1–10 nM, depending on the assays used. In vivo therefore many of the variant stem-loops can be expected to act as PSs. The locations of these variant PSs have been predicted and are shown below on the genetic map of the phage. Those that are also independently identified by the recent cross-linking within infectious phage are highlighted in red.
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f0002: PS-mediated assembly of the T = 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. Binding to TR, and other related stem-loops, triggers a conformational change to the A/B-like dimer. Such RNA switching is required up to 60 times to make the T = 3 shell. (B) There is only a single copy of TR within the MS2 genome but many variant stem-loops can be formed that contain some of the important recognition features for CP31. Note, when these interactions (arrowed in figure) are measured in vitro their affinities are lowered significantly (shown as relative reduced affinities compared to TR). However, the concentration of a single stem-loop in the volume of the capsid is ˜0.3 mM. This compares with the Kd for TR binding to a CP dimer which is 1–10 nM, depending on the assays used. In vivo therefore many of the variant stem-loops can be expected to act as PSs. The locations of these variant PSs have been predicted and are shown below on the genetic map of the phage. Those that are also independently identified by the recent cross-linking within infectious phage are highlighted in red.

Mentions: Single-stranded, positive-sense RNA phages co-assemble their protective coat protein shells around copies of their genomic RNA. This is one of the 2 common virus assembly mechanisms, the other being formation of a procapsid and the subsequent introduction of the viral genome, most commonly in the form of dsDNA, via specialized protein machinery. Spontaneous co-assembly is an essential feature of the lifecycles of a very wide range of important pathogens in every kingdom of life.1 Genome packaging in these viruses has been thought to be dominated by coat protein-coat protein (CP) interactions, and favorable electrostatic contacts that ensure RNA encapsidation, with at most a single, high-affinity CP-binding RNA signal.2-6 Recent work with a variety of viruses in this family suggests that these models are not representative of in vivo scenarios.7 When the first atomic structure of a spherical virus, namely that of Tomato Bushy Stunt Virus (TBSV), was revealed by Harrison and co-workers in a technical tour de force they defined the major structural questions that needed to be answered to explain that structure.8 These are how is genome encapsidation specificity achieved, and secondly how is the quasi-equivalence of the TBSV T=3 capsid established during assembly. We have now provided mechanistic explanations for both these events.9-12 We have shown that multiple RNA sites encompassing minimal CP sequence recognition motifs, when presented via RNA folding, regulate both packaging specificity and the pathway of virion assembly (Fig. 1). In effect, the RNA encodes a set of capsid assembly instructions, via multiple Packaging Signals (PSs). PSs facilitate formation of the protein-protein contacts in their capsids, and for bacteriophage MS2 it has been shown that they also act as allosteric switches determining the formation and placement of the quasi-conformers of the capsomeres forming the T=3 shell13-17 (Fig. 2).Figure 1.


Bacteriophage MS2 genomic RNA encodes an assembly instruction manual for its capsid.

Stockley PG, White SJ, Dykeman E, Manfield I, Rolfsson O, Patel N, Bingham R, Barker A, Wroblewski E, Chandler-Bostock R, Weiß EU, Ranson NA, Tuma R, Twarock R - Bacteriophage (2016)

PS-mediated assembly of the T = 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. Binding to TR, and other related stem-loops, triggers a conformational change to the A/B-like dimer. Such RNA switching is required up to 60 times to make the T = 3 shell. (B) There is only a single copy of TR within the MS2 genome but many variant stem-loops can be formed that contain some of the important recognition features for CP31. Note, when these interactions (arrowed in figure) are measured in vitro their affinities are lowered significantly (shown as relative reduced affinities compared to TR). However, the concentration of a single stem-loop in the volume of the capsid is ˜0.3 mM. This compares with the Kd for TR binding to a CP dimer which is 1–10 nM, depending on the assays used. In vivo therefore many of the variant stem-loops can be expected to act as PSs. The locations of these variant PSs have been predicted and are shown below on the genetic map of the phage. Those that are also independently identified by the recent cross-linking within infectious phage are highlighted in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836477&req=5

f0002: PS-mediated assembly of the T = 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. Binding to TR, and other related stem-loops, triggers a conformational change to the A/B-like dimer. Such RNA switching is required up to 60 times to make the T = 3 shell. (B) There is only a single copy of TR within the MS2 genome but many variant stem-loops can be formed that contain some of the important recognition features for CP31. Note, when these interactions (arrowed in figure) are measured in vitro their affinities are lowered significantly (shown as relative reduced affinities compared to TR). However, the concentration of a single stem-loop in the volume of the capsid is ˜0.3 mM. This compares with the Kd for TR binding to a CP dimer which is 1–10 nM, depending on the assays used. In vivo therefore many of the variant stem-loops can be expected to act as PSs. The locations of these variant PSs have been predicted and are shown below on the genetic map of the phage. Those that are also independently identified by the recent cross-linking within infectious phage are highlighted in red.
Mentions: Single-stranded, positive-sense RNA phages co-assemble their protective coat protein shells around copies of their genomic RNA. This is one of the 2 common virus assembly mechanisms, the other being formation of a procapsid and the subsequent introduction of the viral genome, most commonly in the form of dsDNA, via specialized protein machinery. Spontaneous co-assembly is an essential feature of the lifecycles of a very wide range of important pathogens in every kingdom of life.1 Genome packaging in these viruses has been thought to be dominated by coat protein-coat protein (CP) interactions, and favorable electrostatic contacts that ensure RNA encapsidation, with at most a single, high-affinity CP-binding RNA signal.2-6 Recent work with a variety of viruses in this family suggests that these models are not representative of in vivo scenarios.7 When the first atomic structure of a spherical virus, namely that of Tomato Bushy Stunt Virus (TBSV), was revealed by Harrison and co-workers in a technical tour de force they defined the major structural questions that needed to be answered to explain that structure.8 These are how is genome encapsidation specificity achieved, and secondly how is the quasi-equivalence of the TBSV T=3 capsid established during assembly. We have now provided mechanistic explanations for both these events.9-12 We have shown that multiple RNA sites encompassing minimal CP sequence recognition motifs, when presented via RNA folding, regulate both packaging specificity and the pathway of virion assembly (Fig. 1). In effect, the RNA encodes a set of capsid assembly instructions, via multiple Packaging Signals (PSs). PSs facilitate formation of the protein-protein contacts in their capsids, and for bacteriophage MS2 it has been shown that they also act as allosteric switches determining the formation and placement of the quasi-conformers of the capsomeres forming the T=3 shell13-17 (Fig. 2).Figure 1.

Bottom Line: They act co-operatively to regulate the 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 T=3 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.

View Article: PubMed Central - PubMed

Affiliation: Astbury Center for Structural Molecular Biology, University of Leeds , Leeds, UK.

ABSTRACT

Using RNA-coat protein crosslinking we have shown that the principal RNA recognition surface on the interior of infectious MS2 virions overlaps with the known peptides that bind the high affinity translational operator, TR, within the phage genome. The data also reveal the sequences of genomic fragments in contact with the coat protein shell. These show remarkable overlap with previous predictions based on the hypothesis that virion assembly is mediated by multiple sequences-specific contacts at RNA sites termed Packaging Signals (PSs). These PSs are variations on the TR stem-loop sequence and secondary structure. They act co-operatively to regulate the 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 T=3 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.

No MeSH data available.


Related in: MedlinePlus