Human cytomegalovirus strain-specific differences in protein expression of type I IFN pathway proteins do not impact virus replication
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The type I IFN response is crucial for cells to restrict viral replication during infection. Many viruses, including human cytomegalovirus (HCMV), have evolved mechanisms to antagonize the type I IFN response. We have previously observed an increase in protein expression of certain IFN-stimulated genes when comparing the high-passage HCMV strain AD169 to the low-passage strain HCMV Merlin, suggesting that AD169 is defective in its ability to inhibit type I IFN function. To better understand HCMV interaction with the type I IFN response, we examined expression of cellular and viral proteins expressed in Merlin- and AD169-infected cells associated with IFN production and signalling. HCMV IFN antagonists expressed by both viruses had differences in amino acids throughout their protein sequences, although analysis using AlphaFold revealed that there was likely to be no obvious differences in the overall structure of these proteins. Analysis of quantitative mass spectrometry datasets showed modest differences in the expression of cellular IFN-associated proteins between strains. Contrary to previously reported data, we found no obvious loss of IRF3 expression, though this may be due to experimental differences between studies. These data revealed that multiplicity of infection was an important factor in IRF3 expression. We found little or no statistical difference in the production of IFN- β RNA between Merlin- and AD169-infected cells in reverse transcriptase quantitative PCR assays and little or no statistical difference in replication of AD169 and Merlin in virus replication assays. Overall, these data suggest that different strains of HCMV have different, albeit modest, abilities to influence the expression of type I IFN pathway proteins during infection. However, this had no overall impact on the ability of different strains to produce a type I IFN or to replicate.
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The work is well presented and represents a topic that will be interesting to the field. All of the reviewers comments have been addressed in the revised manuscript and the paper is now ready for publication
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Comments to Author
Overall, I enjoyed reading this manuscript and found the study to be interesting and thoughtfully executed. The work addresses an important question, and with some refinement, its impact and clarity could be further strengthened. In the introduction, the manuscript would benefit from clarifying the motivation earlier by moving the prior observation that Merlin and AD169 differ in ISG responses (particularly MxA and ZAP-S) closer to the beginning, which would more clearly frame the purpose of the study. The re-analysis of published proteomics datasets is a useful addition, and its interpretation could be strengthened by defining what constitutes a biologically meaningful fold change and by acknowledging the inherent limitations of relying on previously published datasets, such as batch effects, …
Comments to Author
Overall, I enjoyed reading this manuscript and found the study to be interesting and thoughtfully executed. The work addresses an important question, and with some refinement, its impact and clarity could be further strengthened. In the introduction, the manuscript would benefit from clarifying the motivation earlier by moving the prior observation that Merlin and AD169 differ in ISG responses (particularly MxA and ZAP-S) closer to the beginning, which would more clearly frame the purpose of the study. The re-analysis of published proteomics datasets is a useful addition, and its interpretation could be strengthened by defining what constitutes a biologically meaningful fold change and by acknowledging the inherent limitations of relying on previously published datasets, such as batch effects, differences in MOI, and other experimental variables. The sequence and structure analyses are appealing, but might benefit from a brief justification for the selection of five high-passage versus five low-passage strains and from moderating statements regarding AlphaFold predictions, which should probably be described as indicative rather than definitive; inclusion of a quantitative structural comparison (e.g., RMSD) would further enhance this section if feasible. The IRF3 experiments would be clearer with additional rationale for the choice of serum starvation, dexamethasone treatment, and MOI as experimental variables, and the proposed "bystander-cell" explanation should be framed as speculative unless directly tested; similarly, the use of β-actin as a loading control at high MOI should be justified. I also recommend avoiding over-interpretation of non-significant differences in IFN-β induction or viral replication by rephrasing conclusions to emphasise the absence of statistically significant differences rather than biological equivalence, and by acknowledging that replication differences observed with Merlin(R1111) may reflect entry or tropism defects associated with UL128-131 deletions. Finally, a number of minor revisions would improve readability and rigor, including tightening long sentences, reducing repetitive wording, correcting typographical and formatting inconsistencies, adding missing methodological details (such as numbers of biological replicates, infection conditions, and plaque assay detection limits), and ensuring that all claims are supported by appropriate and consistently formatted citations.
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?
Strongly 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
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The work is well presented and represents a topic that will be interesting to the field. The reviewers have recommended minor amendments for publication of this work. Please ensure that you address their comments.
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Comments to Author
Latham and colleagues present an interesting analysis of type I interferon expression in response to fibroblast infection of different cell types. They reveal that some findings currently accepted as dogma may be due to high MOI experimental designs in proteomics, rather than intrinsic aspects of HCMV biology. Overall a thorough piece of work. Could the authors please clarity Lines 77-78 - Does AD169 contain these genes? Line 235 - I could not follow the logic of why these functions would be conserved in low passage strains but lost with high passage (surely the more you grow the virus, the more selection there is for increased replication?) Line 340 I also thought that this phenotype may be so conserved that it is in every viable lab strain, ie if this function is lost, the virus is immiediately …
Comments to Author
Latham and colleagues present an interesting analysis of type I interferon expression in response to fibroblast infection of different cell types. They reveal that some findings currently accepted as dogma may be due to high MOI experimental designs in proteomics, rather than intrinsic aspects of HCMV biology. Overall a thorough piece of work. Could the authors please clarity Lines 77-78 - Does AD169 contain these genes? Line 235 - I could not follow the logic of why these functions would be conserved in low passage strains but lost with high passage (surely the more you grow the virus, the more selection there is for increased replication?) Line 340 I also thought that this phenotype may be so conserved that it is in every viable lab strain, ie if this function is lost, the virus is immiediately selected against Line 202 - Typo repeating in the expression Line 312 Typo sing (should be using I assume)
Please rate the manuscript for methodological rigour
Very 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?
Strongly 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
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