PREreview of Flaviviruses alter endoplasmic reticulum-mitochondria contacts to regulate respiration and apoptosis

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: 

Flaviviruses alter endoplasmic reticulum-mitochondria contacts to regulate respiration and apoptosis  

Wesley Freppel, Anaïs Anton, Zaynab Nouhi, Clément Mazeaud, Claudia Gilbert, Nicolas Tremblay, Viviana Andrea Barragan Torres, Aïssatou Aïcha Sow, Xavier Laulhé, Alain Lamarre, Ian Gaël Rodrigue-Gervais, Andreas Pichlmair, Pietro Scaturro, Laura Hulea, and Laurent Chatel-Chaix.doi: https://doi.org/10.1101/2023.03.09.531853.  

  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: Greater understanding of virus-host interactions are required to support the development of therapeutic interventions for arthropod-borne flaviviruses, Dengue virus (DENV) and Zika virus (ZIKV). Chatel-Chaix and colleagues previously reported that DENV infection alters mitochondrial morphodynamics and that DENV NS4B protein causes elongation of mitochondria, which physically contact ER-derived cytoplasmic substructures called convoluted membranes (CMs) [Chatel-Chaix L, et al. Dengue Virus Perturbs Mitochondrial Morphodynamics to Dampen Innate Immune Responses. Cell Host Microbe. 2016 Sep 14;20(3):342-356. doi: 10.1016/j.chom.2016.07.008. Epub 2016 Aug 18. PMID: 27545046; PMCID: PMC7105029]. Mitochondrial elongation compromises the integrity of ER-mitochondria contacts (ERMCs) required to coordinate antiviral responses. In the current study, Freppel, W. et al. investigate flavivirus-induced ERMC perturbations and impacts on processes that involve mitochondria like respiration and apoptosis. They conducted comprehensive ultrastructural analysis of DENV- and ZIKV-infected cells to confirm that infected cells displayed mitochondrial elongation and loss of ERMCs. ERMC loss was measured by proximity ligation assays that monitored the association of three ERMC tethering complexes. Genetic disruption of ERMC contacts by silencing of key ERMC proteins increased viral replication, consistent with the known role of ERMCs in facilitating antiviral responses. Infected cells displayed defects in mitochondrial respiration. Conserved NS4B proteins from ZIKV and DENV were sufficient to recapitulate these mitochondrial defects. Finally, the authors demonstrated that genetically interfering with ERMCs by decreasing levels of key partner proteins suppressed accumulation of executioner caspases in infected cells; accordingly, they conclude that ERMC instability may benefit flaviviruses by apoptosis suppression.

OVERALL ASSESSMENT: The authors used a variety of methods to demonstrate ERMC disruption and downstream functional consequences in DENV and ZIKV infected cells. The TEM imaging of mitochondrial morphology and ER-MC interactions, paired with fluorescence microscopy approaches, demonstrated that infection with either ZIKV and DENV alters physical contacts between the mitochondria and the ER and perturbs mitochondrial morphodynamics. Metabolomic, mitoproteomic, and OCR data clearly demonstrated virus-induced mitochondrial stress and diminished respiration, even though mitochondrial membrane potential was maintained. The authors clearly demonstrated that NS4B is sufficient to elicit these changes in ERMCs and mitochondria.

STRENGTHS: 

·       The authors built upon past studies of flaviviruses and mitochondria to shine a light on ERMCs as a node of control.

·       The use of orthogonal approaches to investigate mitochondrial defects was viewed as a strength of the study. This includes the genetic silencing of ERMC tethering complex components which allowed the authors to conclude that mitochondrial function was not only impaired due to morphodynamic change.

·       We appreciated the use of mitotracker dye to ensure that, despite evidence of mitochondrial stress and diminished respiration, that membrane potential remained intact.

·       We appreciated the large sample sizes studied during ultrastructural analysis which supported quantitative conclusions.

·       The demonstration that NS4B proteins from different flaviviruses are sufficient to cause ERMC instability and mitochondrial dysregulation was viewed as a strength of the study. 

WEAKNESSES:  

·         Uneven writing undermines the impact of this manuscript. The Introduction should be revised for greater clarity and to introduce, and tie together, relevant background and rationale for the study. Some important background information was missing or incompletely described (e.g. NS4B) and there was insufficient introduction of the outstanding questions in the field that are being addressed in this study. Similarly, in the Results section, different experimental approaches are not properly introduced and justified in the context of addressing a research question. One good practice is to begin each new Results section with framing the research question and providing rationale/justification for the experimental approach. For example, such an approach will help the reader appreciate why the authors chose to use mitoproteomics, metabolomics or OCR in different situations to address discrete research questions.

·         The proximity ligation assays in Fig. 2b would benefit from additional specificity controls, as PLA assays are limited by antibody specificity.

·         Differences in the timing for observation/harvest should be justified in the text. For example, TEM experiments concluded at 48 hours post-infection (hpi), whereas PLA experiments concluded at 72 hpi. There may be good reasons for these differences, but they should be justified.

·         The authors nicely paired ZIKV and DENV data throughout the manuscript until Fig. 6, which leaves the reader wondering if DENV also affects apoptosis. Including DENV data in Fig. 6 would provide a pleasing symmetry to the dataset.     

DETAILED U.P. ASSESSMENT: 

OBJECTIVE CRITERIA (QUALITY) 

1.   Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 2

·     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.

·     Fig. 2B: There was consensus that the proximity ligation assays would benefit from additional specificity controls, as PLA assays are limited by antibody specificity. This is required to fully support the claim made in line 171, “This demonstrates a decrease in the abundance of the RRBP1-SYNJ2BP, IP3R1-VDAC1 and VABP-PTPIP51 ERMC tethering complexes and confirms an alteration of ERMCs by DENV and ZIKV at the molecular level.” 

·     Fig. 2D: The RRBP1 western blot is poor quality and does not support the claim that RRBP1 decreases over time. Variability in the actin loading control western blot further undermines this claim. Total protein quantitation (e.g. Stain-free technology) provides an attractive alternative to actin.

·     Are specific analyses performed using methods that are consistent with answering the specific question?  

·     Yes, but as mentioned above, better explanation of the rationale for use of particular experimental approaches would really help the reader. 

·     Is there appropriate technical expertise in the collection and analysis of data presented? 

·     Yes. Clear demonstration of technical expertise throughout. There was only some concern about the quality of western blots in Figure 2.   

·     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?  

·     Yes.

·     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.  

·     Yes. The work is consistent with experimental foundations in the field.

2.   Quality: Completeness (1–3 scale) SCORE =  2

·     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. 

·       The authors used a variety of methods to demonstrate ERMC disruption and downstream functional consequences in DENV and ZIKV infected cells. Overall, the use of orthogonal approaches to investigate mitochondrial defects was viewed as a strength of the study.

·     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. 

·       No fatal flaws in the paper were identified.

3. Quality: Reproducibility (1–3 scale) SCORE = 2

·     Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

·       Fig 1: The ultrastructural data shown was quantification from two independent experiments, but it was justified in text that these experimental groups were large enough to obviate the need for a third independent experiment.

·       Numbers of independent biological replicates (N) should be clearly indicated in all figure legends.

·     Is there sufficient raw data presented to assess rigor of the analysis? 

·       OK

·     Are methods for experimentation and analysis adequately outlined to permit reproducibility? 

·       We were concerned about the lack of rationale/justification for choice of experimental approaches to address discrete questions, as well as the variation in time points, sampling procedures, etc. This undermines reproducibility of the research.

·     If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?  

·       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? 

     Yes

·     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? 

     Yes

·     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. 

·     As described above, uneven writing undermines the impact of this manuscript. The Introduction should be revised for greater clarity and to introduce, and tie together, relevant background and rationale for the study. Some important background information was missing or incompletely described (e.g. NS4B) and there was insufficient introduction of the outstanding questions in the field that are being addressed in this study. Similarly, in the Results section, different experimental approaches are not properly introduced and justified in the context of addressing a research question. One good practice is to begin each new Results section with framing the research question and providing rationale/justification for the experimental approach. For example, such an approach will help the reader appreciate why the authors chose to use mitoproteomics, metabolomics or OCR in different situations to address discrete research questions.

·     Fig 3E: The MitoTracker Orange staining experiments found in the Supplemental Figures establish that membrane potential is largely intact; we think that presenting this data first provides a good foundation for the reader before delving into the mitochondrial respiration measurements in Fig. 3E.  

·     Minor comments:

·     Fig 1C: bar graphs would be easier to interpret if brackets marked the groups being compared for statistical significance.

·     Fig 2A: The text in this cartoon is small and difficult to read.

·     Fig 3F: The heat map data superimposed on a Krebs Cycle/ETC cartoon is complicated and hard to interpret. We suggest that a clearer representation of the heat map alone will really help the reader interpret this data better, whereas the cartoon could be displayed for reference elsewhere.   

·     Numbers of independent biological replicates (N) should be clearly indicated in all figure legends.   

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.] 

·     This manuscript builds upon a very impactful previous paper from Dr. Chatel-Chaix et al. in Cell Host & Microbe in 2016. In a sense, the demonstration of ERMC disruption and mitochondrial dysfunction in cells infected with DENV or ZIKV provides evidence for a broader importance of these discoveries. This manuscript provides a fundamental foundation for understanding this node of control in virus infected cells, which could be applicable for diverse viruses beyond these two flaviviruses.

Impact: Extensibility (1–4 or N/A scale) SCORE =  1.5

·     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)?  

·     No extension to human cohort necessary for this to represent an important advance.

·     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.