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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.

Published onNov 10, 2023
Review 2: "Sequential Membrane- and Protein-bound Organelles Compartmentalize Genomes During Phage Infection"

RR:C19 Evidence Scale rating by reviewer:

  • Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.



The study of Armbruster et al. uncovered the functions of phage-generated organelles in the infection cycle of bacteriophages from the family Chimalliviridae. The representatives of this family are unique among other prokaryotic viruses owing to their ability to form phage nucleus which was previously demonstrated to protect the phage genome from the action of DNA-targeting defense systems of a bacterial host. The authors demonstrated that the proteinaceous phage nucleus is essential for phage DNA replication, while a membrane-bound vesicle protects the phage DNA before the formation of phage nucleus and ensures early phage transcription. 

In this work, using an elegant CRISPRi-ART tool to inhibit the synthesis of phage-encoded protein chimallin (ChmA), a main component of the phage nucleus shell, the authors investigated whether the phage nucleus is required for the phage life cycle. They demonstrated that besides its protection function, the phage nucleus is essential for phage DNA replication. When the phage nucleus was not formed due to the knockdown of ChmA synthesis, phage DNA inside the infected cell remained unamplified in the amount of single genome injected into the cell. Complementation of ChmA restored the formation of phage nucleus and phage replication in knockdown cells. Moreover, phage DNA polymerase responsible for replication of the phage genome was shown to be localized inside the phage nucleus, further confirming its role as a replication compartment.

The authors also showed that immediately upon injection into the cell, phage DNA is located inside a membrane-bound vesicle which protects the phage genome from host DNases before the formation of phage nucleus. These vesicles were referred to as the early phage infection (EPI) vesicles. The authors revealed that phage DNA is actively transcribed in EPI vesicles probably by a virion RNA polymerase responsible for the transcription of early phage genes and injected along with the phage genome in the cell. Another, phage-encoded non-virion RNA polymerase is responsible for late transcription. Mass spectrometry analysis revealed an active phage protein synthesis in the absence of ChmA that further supports that EPI vesicles are transcriptionally active.

Thus, the authors provide multiple lines of evidence of the role of phage nucleus in phage on and the role of EPI vesicle in early phage transcription. However, it is not clear what phage structure ensures later transcription by non-virion RNA polymerase. Given that the authors demonstrated that EPI vesicle is not accessible for replication enzymes, it is tempting to suggest that non-virion RNA polymerase synthesized in the cytoplasm is imported into the phage nucleus to transcribe late genes. However, the results of mass spectrometry showed that many phage proteins were synthesized during ChmA knockdown in the absence of phage nucleus, including virion structural proteins and other products of phage late genes. It would be beneficial to address this concern about late phage transcription.

Overall, the manuscript is clearly written and well-illustrated, the readers can easily follow the results and their interpretations. The conclusions are supported by convincing data of high quality.

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