SARS-CoV-2 ORF8 Exploits Host miRNA Networks to Rewire Post-Transcriptional Regulation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Fernandez-Rodriguez et al explore the impact of SARS-CoV-2 ORF8 over-expression on miRNA regulatory networks using the immortalized A549 lung epithelial cell line. They observe a number of differences between the ORF8 over-expression and control cells, with target validation demonstrating a very modest difference. Overall, this could be an interesting study but given the very modest magnitude of observed changes between ORF8 over-expression and control cells, the lack of strong correlation with previous clinical observations, and the lack of validation using physiologic ORF8 expression levels or infection with the full-length virus, the conclusions drawn within this study can’t be substantiated.

 

 

Major:

• The results section needs to include information regarding how ORF8 was introduced into A549 cells and if the cell population was selected to ensure that all cells express ORF8.

 

• What is the ORF8 expression level in the A549 cells? Is it physiologic compared to levels observed during active infection? An immunoblot should be included that 1) demonstrate the level of ORF8 expression compared to control cells, and 2) demonstrate that the expression level of ORF8 is comparable to a natural infection.

 

• The authors use patient data to justify their findings related to up- or down-regulated miRNAs identified from their over-expression system, however, there is very little overlap between the clinical samples and the data presented herein. The authors claim these differences “suggest that Orf8 may drive previously unrecognized miRNA regulatory networks.” This is an inappropriate conclusion given that they are using an over-expression system in an immortalized cell line.

 

• In addition to the point above, because there is so little overlap between the findings presented herein and with clinically observed changes in patients, the authors need to validate their observations for at least some of the miRNA using full-length SARS-CoV-2 to demonstrate that their results are not due ORF8 over-expression artifacts.

 

• A majority of the expression differences between control and ORF8 over-expression cells are very modest, which is concerning given that this is an over-expression system. If ORF8 were expressed at physiologic levels, would there be an observable difference?

 

Minor:

• Abstract states “A549 cells transduced with ORF8 and wild-type controls” but it is unclear what wild-type controls means in this context. This phrase is repeated throughout the text and should be changed to either “empty vector” or simply “control cells.”

Author Response

Please see the attachment 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors studied how ORF8 expression alters the microRNA landscape of lung epithelial cells and how these changes lead to functional proteomic changes. The study is interesting, but several points require clarification and refinement. Comments are listed below.

1. Lines 52–53. Please describe the mechanism by which ORF8 interferes with MHC-I presentation and the interferon type I signaling pathway. This statement is currently declarative. Adding 1–2 sentences describing the mechanism would significantly enhance the information contained in the introduction.

2. Lines 66–67. This paragraph discusses the roles of miRNAs in immune regulation, inflammation, apoptosis, and epithelial homeostasis. It is also appropriate to mention here the results of a recent in silico study that conducted a systems analysis of microRNA regulatory networks in viral infections and identified microRNAs regulating individual stages of the immune response 10.3390/ijms262010100.

3. Line 97. Please justify the choice of the A549 cell line. Why was this particular line chosen and not, for example, primary respiratory epithelial cultures or the Calu-3 line, which is more physiologically relevant for SARS-CoV-2? Furthermore, was it directly proven that the cells actually express ORF8 at the protein level (Western blot or immunofluorescence)?

4. Line 182. Two studies were selected for comparison with clinical data [42,43]. First, justify the selection of these two studies—why these two and not other available publications on miRNA profiles in COVID-19? Second, two studies are insufficient for a reliable comparison; consider including additional cohorts. Third, provide additional information on the clinical status of the patients in each study: were they hospitalized? Did they require intensive care? Did they survive the infection? What was the spectrum of disease severity? This information is critical for interpreting signature matches, as miRNA profiles can vary significantly depending on disease severity.

5. Discussion Section. The text does not discuss whether ORF8 influences miRNA biogenesis (Drosha, Dicer, DGCR8, Exportin-5). Given that ORF8 causes large-scale reorganization of the miRNA landscape, a discussion of possible mechanisms at the level of the miRNA biogenesis machinery would strengthen the study. Is this due to a direct interaction of ORF8 with components of microRNA biogenesis, or is the effect mediated by transcriptional changes?

6. Limitations Section. The authors use ORF8 from the Wuhan-Hu-1 variant, but ORF8 is one of the most variable proteins in SARS-CoV-2 (including deletions, such as in the B.1.1.529/Omicron variant, and stop codon mutations). While the authors mention this as a direction for future research, a more detailed discussion of the extent to which the findings of this study apply to currently circulating variants in which ORF8 is functionally altered or missing deserves further consideration.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed my previous comments and concerns

Reviewer 2 Report

Comments and Suggestions for Authors

The new additions to the manuscript made a big difference. The quality of the paper had improved, and all my questions were addressed. No more comments.