A transcriptionally active lipid vesicle encloses the injected Chimalliviridae genome in early infection

  1. Emily G. Armbruster
  2. Phoolwanti Rani
  3. Jina Lee
  4. Niklas Klusch
  5. Joshua Hutchings
  6. Lizbeth Y. Hoffman
  7. Hannah Buschkaemper
  8. Eray Enustun
  9. Benjamin A. Adler
  10. Koe Inlow
  11. Arica R. VanderWal
  12. Madelynn Y. Hoffman
  13. Daksh Daksh
  14. Ann Aindow
  15. Amar Deep
  16. Zaida K. Rodriguez
  17. Chase J. Morgan
  18. Majid Ghassemian
  19. Thomas G. Laughlin
  20. Emeric Charles
  21. Brady F. Cress
  22. David F. Savage
  23. Jennifer A. Doudna
  24. Kit Pogliano
  25. Kevin D. Corbett
  26. Elizabeth Villa
  27. Joe Pogliano

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Abstract

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses 1–3 . Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA 4–10 . Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae , but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART 11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

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  1. Version published to 10.1101/2023.09.20.558163v2 on bioRxiv
  2. Rapid Reviews Infectious Diseases

    Kevin Forsberg

    Review 3: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

    Overall, the reviews provide critical scrutiny of the methodology and balanced perspective on the merits and limitations of this preprint's conclusions.

  3. Rapid Reviews Infectious Diseases

    Ekaterina Semenova, Hiba Shaqra

    Review 2: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

    Overall, the reviews provide critical scrutiny of the methodology and balanced perspective on the merits and limitations of this preprint's conclusions.

  4. Rapid Reviews Infectious Diseases

    Maria Yakunina, Daria Antonova

    Review 1: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

    Overall, the reviews provide critical scrutiny of the methodology and balanced perspective on the merits and limitations of this preprint's conclusions.

  5. Rapid Reviews Infectious Diseases

    Strength of evidence

    Reviewers: M Yakunina & D Antonova (Peter the Great St.Petersburg Polytechnic University) | πŸ“—πŸ“—πŸ“—πŸ“—β—»οΈ
    E Semenova & H Shaqra (Rutgers University) | πŸ“˜πŸ“˜πŸ“˜πŸ“˜πŸ“˜
    K Forsberg (University of Texas) | πŸ“˜πŸ“˜πŸ“˜πŸ“˜πŸ“˜

  6. Arcadia Science

    viral mRNAs are exported to the cytoplasm to be translated by host ribosomes

    Based on Figure 1B, it seems like you are suggesting that knockdown occurs after the phage nucleus already forms, but this seems contradictory to rest of the results. So maybe 1B is more of a generic overview of how this knockdown technology is particularly useful in nucleus-forming phage. However, since in this paper the knockdown is presumably happening before nucleus formation, it might be good to have a figure somewhere reflecting that, as I found 1B to be a little confusing in the context of ChmA.

  7. Arcadia Science

    reduced ∼1,000-fold

    I'm curious what the thresholds are for considering something to be essential for phage reproduction? 1000x reduction is huge, but there is still a decent amount of plaquing shown in figure 1F.

  8. Arcadia Science

    the EPI vesicle is transcriptionally active and is a bona fide stage in the Chimalliviridae life cycle

    this is so interesting! I wonder whether the EPI vesicle is found in non-nucleus forming jumbo phage? Is it maybe an intermediate step in the evolution of these phage?

  9. Arcadia Science

    Thus, Chimalliviridae form sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the life cycle.

    Congrats on a really beautiful paper! So much cool new biology!

  10. Version published to 10.1101/2023.09.20.558163v1 on bioRxiv