Effect of CHIKV capsid protein nucleolar localisation on host gene modulation

  1. Shambhavi Rao
  2. Nicholas P. West
  3. Ali Zaid
  4. Suresh Mahalingam
  5. Adam Taylor

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Abstract

Host cell nuclear localisation of alphaviral proteins has been shown to be important for host transcriptional shut off, antagonising type I interferon induction and inhibiting the antiviral response. Our previous studies demonstrate that mutation of the nucleolar localisation sequence (NoLS) of chikungunya virus (CHIKV) capsid protein significantly attenuates CHIKV replication. However, the reason for this attenuation remains unclear. In this study we investigated the impact of CHIKV capsid protein on host gene expression using Nanostring analysis and determined whether nucleolar localisation of capsid protein is required for host gene modulation. Little significant change in differential gene expression (adjusted p value <0.05) was observed in Capsid-WT-EGFP and mutant Capsid-NoLS-EGFP transfected HEK293T cells compared to mock EGFP transfected cells. To explore minor changes to host gene modulation in response to capsid protein, differential gene expression analysis was performed under a reduced arbitrary threshold (unadjusted P value < 0.01). Results suggest that, expressed as a recombinant protein, CHIKV capsid has limited impact on host gene expression, regardless of its ability to localise to the nucleolus. Results further suggest that attenuation of CHIKV resulting from mutation of the capsid protein NoLS is largely dependent on viral rather than host factors.

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  1. Access Microbiology

    Thank you for submitting your paper to Access Microbiology. It has now been reviewed and I would like you to revise the paper in line with the reviewers' reports and any Editorial Office requirements below. The reviewer reports can be found at the bottom of the email. While it is understandable that no additional experimental work can be undertaken and we agree that the limitation of the experiments are clearly stated, I strongly invite you to address the suggestion of the referee in revisiting your REVIGO analysis in Fig4a.

  2. Access Microbiology

    Comments to Author

    The authors have address our comments by addition discussions of the limitations of their study. Except for an important localization control, they have elected not to do additional experiments, which is their prerogative. The one point I would insist on is using REVIGO to collapse the GO term analysis for Fig.4A. To me it is poorly rigorous to say that they have identified 45 differential GO terms when they have only 32 differentially regulated genes. Their statement that REVIGO would take resources seems strange given that REVIGO is a free online tool and that it would take a few hours at most to redo the analysis, if that.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  3. Version published to 10.1099/acmi.0.000701.v2 on Access Microbiology
  5. Access Microbiology

    Comments to Author

    Rao et al. investigated the influence of intracellular Chikungunya capsid expression on gene expression in mammalian cell lines. The authors hypothesized that the nucleolar localization of the viral capsid might be linked to the previously observed transcriptional shut-off. Utilizing Nanostring technology, the study initially revealed no significant changes in gene expression. However, upon lowering their statistical threshold, the authors identified a limited number of up- or down-regulated targets. This study convincingly demonstrates that the nuclear localization of the capsid does not account for modifications in gene expression. Minor changes: Line 132: Could you provide a brief explanation of Nanostring technology and rationale for its use in your study? Line 135: Could you describe the normal ranges? Figure 1: The dot labels overlap, making the figure challenging to read. The dot legend is unclear, and the legend for white dots is missing. Please adjust Figure 1 to fit on a single page. In the legend (line 155), add "unadjusted" before p-value. Figure 2: In Panel A, make the text color for "Downregulated" white for better visibility. In Panel B, increase the font size for better readability, especially for the dot legends, and ensure consistency with the font used in other panels. Line 210: Even with a reduction in your padj to p-value 2. Discuss this observation either here or in the discussion section to highlight the limitations of your findings and the indication that the viral capsid alone does not significantly modify gene expression. Figure 4: In Panel A, rearrange the Volcano plots labels to avoid overlapping. In Panel B, change the text color for downregulated genes to white for better contrast. Standardize the font and size of the figure legend; Panels A, B, and C should have consistent formatting. Line 231: Clarify that the capsid alone (transfected) shows no impact on gene expression, and this may differ in the case of an actual infection.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    In this manuscript Rao et al. examined the effect of the Chikungunya virus (CHIKV) capsid protein on gene expression. Previous work by the authors has shown that the CHIKV capsid protein has a nucleolar localization sequence (NoLS), and that a CHIKV-NoLS mutant virus exhibits attenuated replication in IFN deficient cells, although the reason remains unclear. The authors performed Nanostring-based differential gene expression (DEG) analysis on HEK293T cells transfected with either a WT version of the CHIKV capsid protein, a mutant version lacking the NoLS, or a GFP control plasmid. Overall, they observed no significant DEGs comparing Capsid-WT-EGFP- and CHIKV-NoLS-transfected cells to GFP-transfected cells, suggesting that expression of the capsid protein in isolation does not significantly impact the host transcriptome. Due to the lack of DEGs with standard settings, they perform further analysis with a reduced arbitrary threshold false discovery rate, which resulted in five DEGs known to be involved in virus-host interactions. Given the absence of robust effects on host gene expression, the authors suggest that attenuation of the CHIKV-NoLS is largely dependent on viral rather than host factors. Given the dearth of information about CHIKV capsid, these data are interesting and novel. However, a key limitation of the study is that the Capsid protein expression and Nanostring analysis was performed in HEK293Ts, which are a transformed cell type lacking certain innate immune sensors that could be relevant in the course of infection. Furthermore, the absence of cytokines normally secreted during infection may also mask a host shutoff/immune regulation function of the capsid protein. Therefore, additional experiments could strengthen the authors' conclusions. Major Points: 1) The primary basis for the conclusions of the paper is Nanostring analysis of uninfected HEK293T cells transfected with EGFP, Capsid-WT-EGFP or Capsid-NoLS-EGFP. As CHIKV can infect a variety of cell types in vivo, it would add rigor to repeat their transfection and Nanostring analysis in another more biologically relevant cell type. Minimally, the possibility that CHIKV capsid could have gene regulatory effects in a more relevant cell type should be discussed. Another experiment that would add to the rigor of the paper if transfection efficiency is too low in other cell types, would be pre-treating their HEK293T cells with IFN prior to transfection, then comparing the transcriptomic results of cells pre-treated with IFN to those at a resting state. This is particularly important given the very recent publication of PMID: 38043141, which shows a different effect of CHIKV capsid. 2) In some host shutoff contexts, if there is a global reduction in mRNAs, the change can be masked if one uses normalization programs that assume that most genes will be the same. It may be worth for the authors to validate some RNAs by RT-QPCR using 18S as normalization control to check if this is a possible explanation for the lack of changes. (unless there is evidence that rRNAs are changed by capsid) 3) Even though they have statistically significant changes in Fig. 2, it would be better to only list and discuss the ones where the change in gene expression is at least 1.5x up or down (log2 +/-0.58), to increase the chances that these may be functionally meaningful changes. Similarly for the DAVID analysis in Fig. 3, it seems odd to have more differential regulated classes than proteins. It would be helpful if they used a tool like REVIGO to collapse the classes into a smaller representative set, since they must have a lot of overlap. 4) One of the most upregulated genes in capsid-GFP vs. GFP is IFI16, and it is not upregulated by capsid-NoLS-GFP. This is interesting in light of this paper: PMID: 31423125. The authors may want to comment on it. That said, why does this not come up as a regulated gene in the analysis in Fig. 4? 5) The authors should rephrase lines 245-246 because they cannot exclude that the NoLS is needed for a function that depends on host factor but is not related to gene expression or immune evasion. Minor 1) The authors mention that CHIKV nsP2 is also involved in host shutoff and also localizes to the nucleus. Have the authors tried co-transfection of a fluorescently tagged nsP2 protein with either Capsid-NoLS-EGFP or WT Capsid-EGFP? Does localization of nsP2 change upon co-transfection with Capsid-NoLS-EGFP? 2) In lines 59-61 the authors may want to specify that the comment re: nuclear localization and antagonism of host responses refers to other proteins from CHIKV and capsid in other alphaviruses, not CHIKV capsid. 3) Lines 65-68 - this paragraph is an unconventional way to end the intro. It would be better to summarize the findings. 4) The methods refer to the construct as being kindly provided by the corresponding author (line 73). That needs to be corrected. 5) It would be good if they could add the data on localization of transfected capsid proteins (lines 128-129). 6) Lines 143-144: do the authors have any sense of how the level of capsid in transfection compare to those in infection? And could they mention it here? 7) Lines 232-234: this sentence seems to invalidate the entire premise for the study - could they clarify by mentioning which specific viruses they are referring to?

    Please rate the manuscript for methodological rigour

    Satisfactory

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Partially support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Version published to 10.1099/acmi.0.000701.v1 on Access Microbiology