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  1. 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:

    Poly(rC)-binding protein 1 limits hepatitis C virus assembly and egress. 

    Sophie E. Cousineau, Selena M. Sagan. bioRxiv 2021.02.28.433252; doi: https://doi.org/10.1101/2021.02.28.433252

    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: Cousineau and Sagan explored the role of poly(rC)-binding protein 1 (PCBP1) in the HCV life cycle. Using the HCVcc system in Huh7.5 cells, they found that PCBP1 silencing decreased viral protein and viral RNA accumulation but increased virion production. After dissecting the viral replication cycle with HCVpp, a replication-defective reporter RNA, and assembly-deficient reporter RNAs, the authors concluded that PCBP1 silencing had no impact on HCV entry, translation, and genome replication. By contrast, inhibition of viral RNA synthesis with 2'CMA increased virion production from PCBP1 silenced cells, suggesting that PCBP1 may modulate HCV assembly and/or egress.

     

    OVERALL ASSESSMENT: 

     

    STRENGTHS: The manuscript is concise, well-structured and straightforward. The finding that PCBP1 hinders a late stage in HCV replication is novel.

     

    WEAKNESSES: The precise mechanism of PCBP1 control of HCV assembly/egress was not fully explored in this manuscript. Data interpretation for Fig. 1, as well as the Methods for Figure 3 and Figure 5 may be confusing and require further exposition/clarification (described in detail below). The reader would benefit from additional context and discussion of our current understanding of HCV assembly/egress, as well as the known roles for PCBP1 in the cell.  

     

    DETAILED U.P. ASSESSMENT:

    OBJECTIVE CRITERIA (QUALITY)

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

    ●      Figure by figure, do experiments, as performed, have the proper controls?

    o   Fig.1: We suggest to include more details in the figure legend, such as indicating whether the HCV/actin quantification was normalized to siRNA control (Fig.1D) and spelling out "focus-forming unit (FFU)" (Fig.1E), to make the figure more self-explanatory.

    o   Fig.2: It is not clear whether the authors used Renilla as an internal control here. We suggest that the authors clarify and present normalized data or include the control data when an internal control was used.

    o   Fig.3: In the figure legend, authors stated that cells were electroporated with both RLuc and FLuc RNAs, therefore, fold change could have been calculated. We suggest the authors to include normalized data or explain why the control was not shown. The control may provide an indication of electroporation efficiency between samples.

    o   Fig.4: Yes

    o   Fig.5: Fig.5D shows an increased intracellular virus accumulation rate in PCBP1 silenced cells, which seems contradictory to the decreased viral proteins and RNA accumulation and unaltered intracellular titers in Fig.1. It would be nice if the authors discussed this discrepancy. In addition, we wondered if the results in Fig.5D would be different if the samples were collected beyond 12 h post-treatment.

     

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

    o   Fig.1: We wonder if the viral protein levels were increased in the supernatant? This would further support the hypothesis that decrease of intracellular viral protein accumulation was due to an increased viral secretion.

    o   Fig.2: HCVpp expressing the glycoproteins of JFH-1T might be a better model for the purpose, considering that (1) JFH-1 and the H77 used here belong to different genotypes, and that (2) JFH-1T has adaptive mutations in the E2 coding region.

    o   Fig.4: Quantification of viral RNA using qRT-PCR may be more accurate, and a comparison of replication kinetics between the packaging-defective viruses and the full-length virus could better demonstrate PCBP1's impact on packaging.

    o   Fig.5: Blocking HCV egress using endosomal inhibitors such as U18666A or Bafilomycin-A1 (https://jvi.asm.org/content/84/21/11590) in PCBP1 silenced cells may directly show the impact of PCBP1 on HCV release.

     

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

    o   Yes

     

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

    o   Fig.5C and E: Statistical analyses were not performed.

    o   Fig.5D and F: We suggest the authors to show the raw data in scatterplots and their regression lines, and specify how the accumulation rates were calculated. It would make the data much clearer. 

     

    ●      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    Yes

     

    2. Quality: Completeness (1–3 scale) SCORE = 1.5

    ●      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   In general, the results support the proposed title and abstract. However, how PCBP1 affects HCV assembly or egress is unclear.

     

    ●      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   Given the implications of PCBP1 on the turnover of MAVS, we wonder if these results could be reproduced in a cell line with RIG-I intact, such as Huh7 cells or primary human hepatocytes.

     

    3. Quality: Reproducibility (1–3 scale) SCORE = 1.5

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

    o   Yes

     

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

    o   Fig.2 and 3: We suggest that the authors show the raw data and luciferase controls, or explain why they didn't include it for normalization.

    o   Fig.5D and 5F: We suggest the authors show raw data in scatterplot as mentioned above.

     

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

    o   For the transfection control in "HCV and VSV pseudoparticles (HCVpp and VSVpp)" and "Luciferase assays", the authors mentioned that Dual Reporter Luciferase kit was used, but all data were shown in RLU. We suggest authors clarify this and present normalized data when an internal control was used.

     

    ●      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 = 2

    ●      Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

    o    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?

    o    We feel that more information on HCV assembly/egress and how PCBP1 could affect these processes would be quite helpful for the reader, both in the Introduction and Discussion.

    o    While PCBP1's role in viral replication cycle was mentioned, more introduction on PCBP1's physiological functions / cellular role in hepatocytes and the poly(rC) targets of PCBP1 will also be helpful.

     

    ●      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 to be significant bases for decisions.

    o    Line 62-line 63, the statement of 'a number of cellular proteins and RNAs have been shown to interact with the HCV genome.....' missing citation.

    o    Bar graphs: Perhaps these results could be shown as boxplots.

     

    MORE SUBJECTIVE CRITERIA (IMPACT)

    1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 3

    ●      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.

    o    This study examined the impact of PCBP1 on each step of HCV viral life cycle and proposed a potential role of PCBP1 in modulating HCV packaging and egress. In-depth analyses on how PCBP1 restricts HCV assembly will add to the impact of this study.

     

    ●      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.

     

    2.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)?

    ●      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).

    o    We suggest this study could be extended to another cell line, such as Huh7, or primary human hepatocytes.

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