A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids

  1. Caroline M Boyd
  2. Sundharraman Subramanian
  3. Drew T Dunham
  4. Kristin N Parent
  5. Kimberley D Seed

  • Curated by eLife

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    eLife assessment

    This valuable study reports on the structure and function of capsid size-determining external scaffolding protein encoded by a Vibrio phage satellite. The structural work is of high quality and the presented reconstructions are compelling. The paper offers a substantial advance in the field of phage and virus structure and assembly, with implications for understanding the evolution of phage satellites.

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Abstract

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae , phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli , we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

Article activity feed

  1. Version published to 10.7554/elife.87611.3 on eLife
  2. Version published to 10.7554/elife.87611 on eLife
  3. Version published to 10.7554/elife.87611.2 on eLife
  4. eLife

    eLife assessment

    This valuable study reports on the structure and function of capsid size-determining external scaffolding protein encoded by a Vibrio phage satellite. The structural work is of high quality and the presented reconstructions are compelling. The paper offers a substantial advance in the field of phage and virus structure and assembly, with implications for understanding the evolution of phage satellites.

  5. eLife

    Reviewer #1 (Public Review):

    This paper describes the discovery, functional analysis and structure of TcaP, a protein encoded by the Vibrio phage satellite PLE, that forms a size-determining scaffold around PLE procapsids made from helper phage ICP1 structural proteins.

    The system displays a fascinating similarity to the P2/P4 system, which had previously been unique in its use of a dominant, size-determining external scaffolding protein (Sid). An interesting observation is that PLE appears to be dependent on small capsids for efficient transduction, a phenomenon not previously seen in headful packaging phage/satellite pairs. It is not clear why this is the case.

    The work is interesting, comprehensive and of high quality. The reconstruction and modeling statistics are good; unfortunately, although the map has clear alpha-helical density around the threefold axes, the TcaP model does not include this critical region. The comparison to Sid provides an illustration of probable convergent evolution.

    The paper constitutes an important contribution to the field of phage and virus structure and assembly, with implications for understanding the evolution of phage satellites and for macromolecular assembly processes in general.

  6. eLife

    Reviewer #2 (Public Review):

    Phage satellites are fascinating elements that have evolved to hijack phages for induction, packaging, and transfer, promoting their widespread dissemination in nature. It is remarkable how different satellites use conserved strategies of parasitism, utilising unrelated proteins that perform similar roles in their cognate elements. In the current manuscript, Dr. Seed and coworkers elucidated the mechanism used by one family of satellites, the PLEs, to produce small capsids, a process that inhibits phage reproduction while increasing PLE transmission. The work is presented beautifully, and the results are astonishing. The authors identified the gene responsible for generating the small capsids, characterised its role in the PLE transfer and phage inhibition, and determined the structure of the PLE-sized small capsids. It is a truly impressive piece of work.

  7. eLife

    Reviewer #3 (Public Review):

    The manuscript by Boyd and co-authors "A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodelling hijacked phage coat proteins into small capsids" reports important results related to self-defending mechanisms that bacteria are used against phages that infect them. It has been shown previously that bacteria produce phage-inducible chromosomal island-like elements (PLE) that encode proteins that are integrated into bacterial genome. These proteins are used by bacteria to amend the phage capsids and to create phage-like particles (satellites) that move between cells and transfer the genetic material of PLE to another bacteria. That study highlights the interactions between a PLE-encoded protein, TcaP, and capsid proteins of the phage ICP1.

    The manuscript is well written, provides a lot of new information and the results are supported by biochemical analysis.

  8. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/10075630.

    Université Toulouse Paul Sabatier, Master Microbiologie Moléculaire

    LMGM-CBI, Toulouse

    This Pre Peer Review is an exercise performed by Master 2 students from University Toulouse Paul Sabatier (Master Microbiologie Moléculaire). Writing a pre-review from a bioRxiv article is part of their module "Scientific analysis".

    It tooks us 3 sessions to finalize the peer-review:

    Session 1: presentation of the scientific edition landscape + article reading

    Session 2: critical reading of each figure

    Session 3: pooling students peer-reviews

    The final text was slightly edited for grammar and syntax but the peer-review reflects the work of the students.

    This manuscript entitled "A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids" from Boyd and co-workers describes how the TcaP protein from the PLE satellite phage can replace the scaffold protein of the ICP1 host phage to form smaller capsids that are appropriate for the PLE genome, and by doing so prevents further ICP1 infections. The authors use an elegant and powerful genetic strategy to obtain escape ICP1 phages that are immune to TcaP, and they use these escapers to further characterize the role of TcaP. They present cryo-microscopy reconstruction structures of the PLE capsids with apparent external scaffold where they are then able to dock alphafold predictions of the capsid and TcaP proteins. The evolution of TcaP proteins from PLE satellites is then examined using old and new environmental isolates of PLE5.

    The materials and methods are in general well explained in great and sufficient detail.

    Legends including gene name, accession and Molecular Weight were greatly appreciated.

    Major Issues :

    • (1) There are no statistical tests for figures 1E, 2D, 5E and 5F.

    • (2) Regarding Figure 2 : 

    No quantification of ICP1 or PLE virions under the conditions shown in Figures 2A to 2C, fig 5. It would be preferable to have a small graph (potentially in supplementary data) with quantifications of each virion type. Currently the reader only has access to the few fields of the electron micrographs chosen by the authors to present in the manuscript.

    Minor Issues :

    • (1)  Regarding Figure 1:

    (1.1) For non experts it is quite enigmatic what big size round shapes represent, or what seems to be double contracted tails. Is it possible to better annotate this panel? same for Figure 2

    (1.2) Panels A and B: It seems there is a lot of cell debris in panel A and very little in panel B. Is that a general difference between the two types of virion preparations or is it specific to the fields chosen for this figure? Perhaps a comment on this from the authors will help the readers.

    (1.3) Panel D: What are the particles of virion size but with black interiors?

    (1.4) We found no details on the discovery of TcaP, even though PLE codes for no known homolog of a protein whose function is homologous to TcaP. Out of curiosity, how did the authors stumble upon TcaP as a candidate?

    (1.5) No access to the source data used to create figure 1E and figure 2D, although it is stated so in the material and methods.

    • (3)  Regarding Figure 3:

    (3.1) There is a contrast between the figure 3 A-2 and 3 A-4,  is the difference in color caused by the presence (or not) of the "decoration" gene ? 

    In contrast to the other fields, Figure 3 A-4 shows white capsids. We would like the authors opinion on this.

    (3.2) In the figure 3B-4, we are not able to see ICP1's scaffold band with the coat on the SDS-PAGE. We would again ask the authors to comment on this.

    (3.3) In figure  3B, a band is visible at the bottom of the SDS Page. Is it an artefact ? Something degraded by a protease in the figure 3-A ?

    (3.4) It would be nice to have a quantification of ICP1 vs PLE sized procapsids for each conditions. Something like a red/blue/grey percentage scale (ICP1/PLE/spiral) next to every platforms to compare each of them, especially for A2-3-5.

    (3.5) Regarding Figure 3 supplements: 

    On Figure 3S1, the chosen axis scale on the second image prevents to see differences between non outlier values

    Figure 3S3 isn't discussed or cited. 

    Figure 3S4 labeling should be reshaped in accordance to the legend, and the figure in itself is quite difficult to read

    • (4)  Regarding Figure 4:

    (4.1) There is a small typography problem on Figure 4's legend line 2: "ICP1's cot",probably meaning  "ICP1's coat" (A few typos in the mat/met as well)

    (4.2) It would be nice to have a representation of how ICP1 scaffold works to compare it with the TcaP's mechanism shown here.

    (4.3) Adding the FSC treshold as a dotted line on the first supplementary figure 4 could be interesting. 

    (4.5) Based on the AA proximity predictions, what about testing particle sizes for an inactive TcaP mutant (R133 for example) ?

    • (5)  Regarding Figure 5:

    (5.1) Numbers for AA and nt would help the readers to orientate themselves on the panel B

    (5.2) For panel E, the lane of pEV isn't shown even though the comparison was done with it.

    (5.3) For panels C, D E and F, which version of PLE5 is used (since the most recent is as functional as PLE1) ?

    (5.4) There is no material and methods for the bioinformatic analyses.

    • (6)  Regarding Figure 6:

    (6.1) Since some escape phages were observed, maybe they could be added to the model, along with their frequency

    (6.2) Questions and perspectives: Out of curiosity, one could ask about the actual length of packages in the different capsids, is it something that is currently under investigation?

    Positive feedback

    (1) Regarding Figure 1:

    Honest about the correctness of the mutant construction: supplementary data with sequencing results

    (2) Regarding Figure 2:

    The presentation of the results is clear

    (3) Regarding Figure 3:

    Clear and comprehensible presentation overall, legends and figures are well though.

    Investigation of the bands obtained in figure 3A-2 in a supp data (Figure 3S2A) to confirm it was protease cleavage is a nice addition. (except the suspicious one cf minor issues of figure 3B)

    (4) Regarding Figure 4:

    Material and methods are quite detailed

    (5) Regarding Figure 5:

    The genetic analysis looks complete.

    Figures are easily readable. The choice of colors helps with the clarity.

    (6) Regarding Figure 6:

    Very clear model.

    Competing interests

    The author declares that they have no competing interests.

    Competing interests

    The author declares that they have no competing interests.

  9. Version published to 10.1101/2023.03.01.530633v2 on bioRxiv
  10. Version published to 10.7554/elife.87611.1 on eLife
  11. eLife

    Author Response:

    We thank the reviewers for their careful and overall positive assessment of our work.

    Reviewer #1 (Public Review):

    This paper describes the discovery, functional analysis and structure of TcaP, a protein encoded by the Vibrio phage satellite PLE that forms a size-determining scaffold around PLE procapsids made from helper phage ICP1 structural proteins. The system displays a fascinating similarity to the P2/P4 system, which had previously been unique in its use of a size-determining external scaffolding protein, Sid. The work is interesting, comprehensive and of high quality. The presentation could be improved as listed in the suggestions below.

    An interesting observation is that PLE appears to be dependent on small capsids for efficient transduction. This is not completely surprising if the element uses a cos site type mechanism for packaging, since this requires an integer number of genomes to be packaged when the capsid is full, and this might be more difficult to accomplish when the helper capsid is much larger than the satellite, as is the case with ICP1. The authors mention in a few places that this is the first known satellite to have this requirement. However, this is not quite correct: a similar defect was seen in phi12/SaPIbov5, where the large phi12 capsid was not quite the right size for either two or three copies of the wildtype ("unevolved") SaPIbov5 (Carpena et al. 2016).

    We thank the reviewer for bringing up this point. First, we agree that for cos type packaging systems, this would not be surprising. However, ICP1 is a pac type phage and we have evidence that PLE is also a pac rather than a cos type packaging satellite; therefore, PLE is the first headful satellite to show such a defect. For cos packaging elements, both SaPIbov5 and P4, non-integer genome lengths have been shown to pack less efficiently into capsids as pointed out above and shown in Carpena et al 2016 and Shore 1978. However, in both of these cases, the genomes were manipulated to change their size, suggesting that naturally occurring cos satellites maintain their genome sizes to be proportional to their capsid sizes or in integer proportion to their helper capsids. We will include a short summary of these previous findings in the main text to provide context for the rare decreases in transduction efficiency reported in the cos satellites.

    The authors present several micrographs showing capsids formed in the presence or absence of wildtype or mutant TcaP and CP (Fig. 1, Fig 2., Fig 3). However, each micrograph shows only a handful of particles of the "correct" size, in addition to a few shells that are aberrant or of a different size. I miss a more statistically rigorous enumeration of shells of different size (PLE or ICP1 sized, or different), empty vs. full, aberrant shells etc. This could be presented as a size distribution graph, a histogram or in table form.

    We thank the reviewer for this recommendation and agree that it would add to the manuscript. We will quantify these particles and present the data in the main text.

    In the abstract, the term "divergent satellite P4" is vague and unclear. Divergent from what? Probably they mean distinct from or unrelated to PLE. Please clarify.

    Yes, we did mean unrelated to PLE, and we will clarify in the text.

    How do they know that gp123 is a decoration protein? Was this previously determined, does it have (sequence) similarity to other known decoration proteins, or is it simply the most likely designation based on its position in the genome?

    Gp123 was annotated based on its position. While there is sequence similarity to other annotated Vibrio phages’ decoration proteins, we will clarify in the text that Gp123 is a putative decoration protein.

    Although the reconstruction and modeling statistics are good, it is difficult to assess the quality of the map and the model from the presented figures. Details of the density and FSC curves (half-map and model-to-map) should be shown. It is also difficult to see the TcaP structure and how it compares to Sid from the figures presented.

    We will address this concern in the revised manuscript.

    Introduction, Paragraph 3: "...which is the number of coat proteins divided by 60" is not strictly speaking the definition of T number. The T number corresponds to the number of subtriangles that one triangular face of the icosahedron is divided into. It corresponds to the number of coat proteins divided by 60 in the canonical case, but in tailed phages, 5 copies are removed to make way for the portal protein. (Other viruses could be described as having architecture corresponding to a specific T number, but with divergent numbers of subunits, e.g. adenoviruses or polyomaviruses.)

    We agree that our simplified explanation of the T number is not entirely accurate and will modify the sentence appropriately.

    Reviewer #2 (Public Review):

    Phage satellites are fascinating elements that have evolved to hijack phages for induction, packaging, and transfer, promoting their widespread dissemination in nature. It is remarkable how different satellites use conserved strategies of parasitism, utilising unrelated proteins that perform similar roles in their cognate elements. In the current manuscript, Dr. Seed and coworkers elucidated the mechanism used by one family of satellites, the PLEs, to produce small capsids, a process that inhibits phage reproduction while increasing PLE transmission. The work is presented beautifully, and the results are astonishing. The authors identified the gene responsible for generating the small capsids, characterised its role in the PLE transfer and phage inhibition, and determined the structure of the PLE-sized small capsids. It is a truly impressive piece of work.

    We thank the reviewer for their positive evaluation of our work.

    Reviewer #3 (Public Review):

    The manuscript by Boyd and co-authors "A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodelling hijacked phage coat proteins into small capsids" reports important results related to self-defending mechanisms that bacteria are used against phages that infect them. It has been shown previously that bacteria produce phage-inducible chromosomal island-like elements (PLE) that encode proteins that are integrated into bacterial genome. These proteins are used by bacteria to amend the phage capsids and to create phage-like particles (satellites) that move between cells and transfer the genetic material of PLE to another bacteria. That study highlights the interactions between a PLE-encoded protein, TcaP, and capsid proteins of the phage ICP1.

    The manuscript is well written, provides a lot of new information and the results are supported by biochemical analysis.

    We thank the reviewer for their supportive evaluation of our work.

  12. eLife

    eLife assessment

    This valuable study reports on the structure and function of capsid size-determining external scaffolding protein encoded by a Vibrio phage satellite. The structural work is of high quality and the presented reconstructions are compelling, but some of the experiments could benefit from a more rigorous statistical analysis of capsid sizes and shapes. The paper offers an advance in the field of phage and virus structure and assembly with implications for understanding the evolution of phage satellites.

  13. eLife

    Reviewer #1 (Public Review):

    This paper describes the discovery, functional analysis and structure of TcaP, a protein encoded by the Vibrio phage satellite PLE that forms a size-determining scaffold around PLE procapsids made from helper phage ICP1 structural proteins. The system displays a fascinating similarity to the P2/P4 system, which had previously been unique in its use of a size-determining external scaffolding protein, Sid. The work is interesting, comprehensive and of high quality. The presentation could be improved as listed in the suggestions below.

    An interesting observation is that PLE appears to be dependent on small capsids for efficient transduction. This is not completely surprising if the element uses a cos site type mechanism for packaging, since this requires an integer number of genomes to be packaged when the capsid is full, and this might be more difficult to accomplish when the helper capsid is much larger than the satellite, as is the case with ICP1. The authors mention in a few places that this is the first known satellite to have this requirement. However, this is not quite correct: a similar defect was seen in phi12/SaPIbov5, where the large phi12 capsid was not quite the right size for either two or three copies of the wild-type ("unevolved") SaPIbov5 (Carpena et al. 2016).

    The authors present several micrographs showing capsids formed in the presence or absence of wildtype or mutant TcaP and CP (Fig. 1, Fig 2., Fig 3). However, each micrograph shows only a handful of particles of the "correct" size, in addition to a few shells that are aberrant or of a different size. I miss a more statistically rigorous enumeration of shells of different size (PLE or ICP1 sized, or different), empty vs. full, aberrant shells etc. This could be presented as a size distribution graph, a histogram or in table form.

    In the abstract, the term "divergent satellite P4" is vague and unclear. Divergent from what? Probably they mean distinct from or unrelated to PLE. Please clarify.

    How do they know that gp123 is a decoration protein? Was this previously determined, does it have (sequence) similarity to other known decoration proteins, or is it simply the most likely designation based on its position in the genome?

    Although the reconstruction and modeling statistics are good, it is difficult to assess the quality of the map and the model from the presented figures. Details of the density and FSC curves (half-map and model-to-map) should be shown. It is also difficult to see the TcaP structure and how it compares to Sid from the figures presented.

    Introduction, Paragraph 3: "...which is the number of coat proteins divided by 60" is not strictly speaking the definition of T number. The T number corresponds to the number of subtriangles that one triangular face of the icosahedron is divided into. It corresponds to the number of coat proteins divided by 60 in the canonical case, but in tailed phages, 5 copies are removed to make way for the portal protein. (Other viruses could be described as having architecture corresponding to a specific T number, but with divergent numbers of subunits, e.g. adenoviruses or polyomaviruses.)

  14. eLife

    Reviewer #2 (Public Review):

    Phage satellites are fascinating elements that have evolved to hijack phages for induction, packaging, and transfer, promoting their widespread dissemination in nature. It is remarkable how different satellites use conserved strategies of parasitism, utilising unrelated proteins that perform similar roles in their cognate elements. In the current manuscript, Dr. Seed and coworkers elucidated the mechanism used by one family of satellites, the PLEs, to produce small capsids, a process that inhibits phage reproduction while increasing PLE transmission. The work is presented beautifully, and the results are astonishing. The authors identified the gene responsible for generating the small capsids, characterised its role in the PLE transfer and phage inhibition, and determined the structure of the PLE-sized small capsids. It is a truly impressive piece of work.

  15. eLife

    Reviewer #3 (Public Review):

    The manuscript by Boyd and co-authors "A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodelling hijacked phage coat proteins into small capsids" reports important results related to self-defending mechanisms that bacteria are used against phages that infect them. It has been shown previously that bacteria produce phage-inducible chromosomal island-like elements (PLE) that encode proteins that are integrated into bacterial genome. These proteins are used by bacteria to amend the phage capsids and to create phage-like particles (satellites) that move between cells and transfer the genetic material of PLE to another bacteria. That study highlights the interactions between a PLE-encoded protein, TcaP, and capsid proteins of the phage ICP1.

    The manuscript is well written, provides a lot of new information and the results are supported by biochemical analysis.

  16. Version published to 10.1101/2023.03.01.530633v1 on bioRxiv