Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories
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Abstract
Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7737272.
We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:
Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories
Jingru Fang, Guillaume Castillon, Sebastien Phan, Sara McArdle, Chitra Hariharan, Mark H. Ellisman, Ashok A. Deniz, Erica Ollmann Saphire. doi: https://doi.org/10.1101/2022.12.27.522024 We will adhere to the Universal Principled (UP) Review guidelines proposed in:
Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7737272.
We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:
Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories
Jingru Fang, Guillaume Castillon, Sebastien Phan, Sara McArdle, Chitra Hariharan, Mark H. Ellisman, Ashok A. Deniz, Erica Ollmann Saphire. doi: https://doi.org/10.1101/2022.12.27.522024 We will adhere to the Universal Principled (UP) Review guidelines proposed in:
Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.
SUMMARY: Many fundamental aspects of Ebola virus (EBOV) replication remain unknown. Here, the authors sought to better understand the role of EBOV viral factories (VF) in spatially regulating viral RNA synthesis. They reconstituted VFs using viral proteins with properties often associated with phase separation (self-oligomerization, RNA binding) including nucleoprotein (NP), polymerase cofactor VP35, and polymerase (L). Condensates were observed in cells transfected with VP35 alone, VP35/NP, or VP35/NP/L. These condensates displayed composition-dependent viscoelastic behavior typical of VFs. Interestingly, L was found to cluster in foci that were interconnected but the distance between the foci was dependent on RNA replication (provided by a minigenome (MG) reporter). VFs formed a typical droplet-like morphology as well as a distinct network-like morphology. To determine if both VF morphologies were present during infection, the authors performed infections with a VP30-deficient EBOV in Vero cells that provide VP30 in trans. They determined that most VFs display droplet-like morphology, but network-like morphology is still present about 30% of the time. Continuing with their transfection-based reconstitution system, the authors designed a split APEX2 (sAPEX2) system to monitor interactions between L and VP35. TEM confirmed the network-like morphology of VFs and localization of L at the periphery of these structures. Finally, using the sAPEX2 system, they utilized four-tilt electron tomography (ET) to resolve 3D images of organelles. Using ET, they obtained clear resolution of the reconstituted VFs and observed that they were close to membrane bound organelles, consistent with previous observations of VFs from other viruses. Interestingly, they also reported loosely coiled structures which they proposed are viral ribonucleoproteins, however this requires further investigation. Together, Fang J., et al. demonstrated the utility of their transfection-based approach for studying EBOV and advanced understanding of EBOV VFs, describing a network-like morphology and composition-dependent distribution of L within VFs.
OVERALL ASSESSMENT: This preprint provides advances understanding of EBOV VFs as biomolecular condensates, providing insight into the interactions, viscoelastic properties, and spatial organization of VF constituents. The study featured cutting-edge microscopy techniques. This study could be strengthened by the addition of key controls, especially for the sAPEX2 system. We also suggest adjustments to the writing to better highlight important findings.
STRENGTHS: This study addresses important questions about the fundamental nature of VFs. The writing was generally clear and accessible, with clear communication of research goals and approaches throughout. For example, the clear Introduction and accompanying methods diagram was very helpful for readers with limited virology knowledge. Without question, the advanced microscopy approaches are impressive, and provide a roadmap for future studies. We appreciated the authors cautious assessment of their findings, which acknowledged experimental limitations and did not overinterpret results. Overall, conclusions were well supported by the data throughout.
WEAKNESSES: We identified the following weaknesses:
1. While we appreciated the power of the sAPEX2 assay to map localization of protein complexes, we think that it could be improved by adding specificity controls. The specificity of the mapping of L+VP35 could be better appreciated if it could be compared to controls (e.g. viral proteins that lack condensate properties and do not participate in VFs).
2. The authors should double-check figure callouts throughout the paper as there are several occasions where the figure they are referring to does not relate to the claim in the sentence. This is a minor weakness, as the data is usually somewhere in the paper, but the misleading callouts were frustrating.
3. Another minor weakness involved the descriptive terminology used in the paper such as "gel-like" or "homogeneous". The authors should define these terms clearly so the reader can understand how they are being used in the context of this study.
DETAILED U.P. ASSESSMENT:
OBJECTIVE CRITERIA (QUALITY)
1. Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.25
● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this 'figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.
● Figures are generally attractive and informative, and the data supports the authors' conclusions. Relatively minor critiques as follows:
o Figure 1: The figure legend has an error, as the descriptors for A and B are reversed. We encourage the authors to review figure callouts in the text as, as there are mismatches. They also refer to Figure 1e (line 165) and it is unclear how that figure supports the claim in the text. We think it would be more appropriate to reference Supplemental Figure 1d along with Figure 1e to support the claim.
o Figure 2: To improve the continuity of Figure 2C, we suggest the authors improve the alignment of the x-axis of the graph with the lanes of the corresponding western blot below. We suggest straight labels or dotted lines to clearly indicate the continuity from the graph to the lanes of the blot. We also suggest increasing the signal of the anti-FLAG blots to give a more obvious positive signal and reveal any additional bands that are currently obscured.
o Figure 3: We suggest clearly indicating which panel shows the network-like or droplet-like phenotypes in Figure 3b. We propose direct labeling on the figure or referencing left and right panels in the figure legend.
o Figure 4: It is unclear what "1" and "2" refer to in the labeling of Figure 4d; we recommend clarifying this on the figure or in the figure legend. It would also be beneficial to include Z-stacks for Figures 4e and 4f to allow the reader to better locate L-VP35-sAPEX2. We also identified a significant error in the interpretation of the data in Figure 4b and 4d. In the text it reads "L-sEX was also expressed to higher levels than wild type L, which could explain the correspondingly higher MG activity seen for sAPEX2-tagged EBOV polymerase (Figure 4d)" however, Figure 4d only shows the expression levels. Furthermore, we do not understand this claim as the MG activity shown in Figure 4b revealed that L-VP35-sAPEX2 had lower activity compared to L-WT + VP35-V5. We encourage the authors to revise this section of text.
o Figure 5: For consistency, a scale bar should be included in the OsO4 panel of Figure 5b. Our more substantial concern is the lack of controls for the sAPEX2 assay. While we acknowledge that this is an innovative tool, it would be more convincing to see the labeling of something that is not their target, also in the phase condensate, which has a different distribution. We suggest potentially using the sAPEX2 assay with NP and VP34. The use of this control would better validate their assay since their observations of the network-like structures on the periphery of VFs seem to contradict the IF data from figure 4.
● Are specific analyses performed using methods that are consistent with answering the specific question?
o We have concerns about the use of the mini-genome as the substrate for polymerase L and indicator of viral replication and transcription within VFs/condensates. Since condensate formation is heavily impacted by the stoichiometry of its constituents, we are concerned that the use of this mini-genome may influence phase separation more than the authors have anticipated. We would also like the authors to discuss the potential implications of the higher expression of VP35 APEX compared to the VP35-V5 construct. Finally, we suggest that the authors use RNA FISH staining to visualize viral RNA to further enhance confocal microscopy experiments.
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● Is there appropriate technical expertise in the collection and analysis of data presented?
o The sAPEX2 construct impacted the intracellular localization pattern of VP35 and induced the formation of network-like VFs. Although the authors showed that co-expression of VP24 and VP35-V5 could also induce the network-like VFs, there are still concerns about potential artefacts caused by sAPEX2 tags. While potentially beyond the scope of this excellent paper, we suggest the authors consider supporting their data with alternative labeling techniques such as immunogold labeling or metal-tagging. The immunogold labeling and metal-tagging have their own limitations, but those techniques do not create extra intermolecular interactions between L and VP35, therefore eliminating potentially artefactual effects on VF morphology. Based on the information in the Results section, the sAPEX2 tagging only allows the authors to examine the network-like VFs with TEM but not droplet-like VFs. It would be very interesting to visualize the droplet-like VPs and the corresponding VP35 distribution. Would the distribution of the VP35 in two phases of VFs align with the IF data? What is the structural/component differences between the network-like and droplet-like VFs? This may provide additional evidence to support the proposed model in the Discussion.
● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?
o OK.
● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.
o OK.
2. Quality: Completeness (1–3 scale) SCORE = 1
● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.
o OK.
● Are there experiments or analyses that have not been performed but if ''true'' would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ''completeness'' here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.
o N/A.
3. Quality: Reproducibility (1–3 scale) SCORE = 1.25
● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?
o While we recognize generating the confocal microscopy data is a cumbersome task, we would like to see three biological replicates for the data presented in Figure 3.
● Is there sufficient raw data presented to assess rigor of the analysis?
o OK.
● Are methods for experimentation and analysis adequately outlined to permit reproducibility?
o Excellent throughout.
● If a ''discovery'' dataset is used, has a ''validation'' cohort been assessed and/or has the issue of false discovery been addressed?
o N/A
4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5
● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?
o OK.
● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?
o Concepts first covered in the Introduction should be revisited in the Discussion to "close-the-loop" and make for more satisfying reading. Please include citations when discussing knowledge gaps and cite other studies that demonstrate why it is important to address these knowledge gaps. Without this, the reader is left with little to refer to other than the Introduction.
● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions.
o We recommend the authors use summary statements as headings in the Results, which would aid reader comprehension throughout. Since the authors created useful methodology diagrams, we suggest the inclusion of a diagram for their proposed model, even though some aspects of the model remain speculative.
MORE SUBJECTIVE CRITERIA (IMPACT)
1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.5
A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ''1'' would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.]
● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.
o The novelty of this study is two-fold; the findings address key knowledge gaps and contribute to a better understanding of Ebola virus VFs, and the validation of the transfection-based minimal system for studying VF condensate properties provides a valuable guide for others. Using the transfection-based model, they observed the contribution of individual viral constituents to VFs and condensate formation. Specifically, they found a reduction in the molecular exchange rate in VFs composed of NP and VP35 in the presence of L. Additional observations included the potential for NP to act as a scaffold in VFs and the influence of viral RNA synthesis on polymerase localization. The authors suggest that these discoveries can be applied to other non-segmented, negative sense RNA viruses. These impressive discoveries might not be fully appreciated by the non-expert reader without further exposition. As suggested in the "scholarship" critique above, revisiting and reinforcing key concepts and providing impact statements in the Discussion may help in this regard.
Impact: Extensibility (1–4 or N/A scale) SCORE = N/A
● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? N/A
● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn't apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ''repeat a mouse-based experiment in humans'') and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). N/A
Competing interests
The author declares that they have no competing interests.
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