Peer review of: Dynamic remodelling of the human host cell proteome and phosphoproteome upon (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

Giansanti et al. - Dynamic remodeling of the human host cell (phospho)proteome upon enterovirus 
infection 

 

GENERAL COMMENTS 

 

This manuscript explores the altered proteome in cells infected with Coxsackievirus B3. The authors 
use MS proteomics to assess the change in the proteome and phosphoproteome in infected cells. 
They find phosphoproteomic changes early in infection, while proteomic changes are only detected 
in later timepoints (after ~8 hrs). The authors use the patterns of phosphorylation to predict the 
kinases involved and investigate their effects on the mTOR pathway specifically. Through 
investigation of the downstream targets of mTOR, they show that eEF2 phosphorylation is decreased 
over the course of infection. The authors also discuss the role of mTOR target TFEB, a regulator of 
lysosomal and autophagy. They find that TFEB is activated by infection and that knockdown of this 
factor inhibits virus production, but not replication, suggesting a role for lysosomal/autophagic 
pathways in egress. 

 

The work reported in the manuscript is well done and describes the phosphoproteomic flux 
associated with infection, which is sure to be a useful reference and source for hyptheses for others 
in the field. The paper itself focuses on mTOR and its downstream targets. These are known to be 
involved in enterovirus biology, as the authors note, which makes their observation less novel, but 
still provides more context for mTOR involvement. The authors suggest one downstream target 
(TFEB) of mTOR that effects virus production, but again this observation lacks mechanistic insight 
beyond the genetic experiment. 

 

Because of its quality, I believe the paper does merit acceptance and publication after the authors 
address some of the comments below. 

 

Specific Comments: 

 

ll. 152-5. “While a decrease in protein levels may be explained by the virus-induced shutdown of 
protein translation, the observation that certain protein 

154 levels increased suggests that a subset of cellular proteins escape the bulk translational 
shutdown induced by viral cleavage of eIF4G.“ Can the author distinguish between altered turnover 
vs. EIF4G independent translation? Any analysis that could be performed to confirm this hypothesis? 

 

Supp. Table 4.3: Why do the authors include Apoptotic signaling in the translation and transcription? 
Perhaps labeling the top enriched categories on the plots would be useful for transparency. 

 

Have the authors considered a gene set enrichment approach based on fold-change of factors in 
given pathways. e.g. Do genes in this pathway have a greater fold-change than expected by chance, 
this might better reflect the phenotype of the infected cells. 

 

Small Notes: 

 

I recommend renaming the section called “Regulation of viral protein levels during CVB3 expression” 
as the section does not discuss regulation perse. 

 

Coxsackievirus is capitalized as it is named after a city “Coxsackie, NY, USA” 

 

Figures: The Blue gradient background is distracting and unnecessary, makes the grey lines and blue 
boxes hard to visualize. 

 

I’d encourage the authors to include some of the more “systems level” figures in the main text to 
give a general sense of the hits on the screen (S2C specifically). 

 

 

Reviewer #2 (Remarks to the Author): 

 

In the manuscript “Dynamic remodeling of the human host cell (phospho)proteome Upon 
enterovirus infection” Giansanti et al present a comprehensive proteomic and phospoproteomic 
dataset across a time course of enterovirus infection. The extensive datasets are analyzed to identify 

changes on the protein and phosphorylation levels and described globally. To interpret findings in 
the phosphoproteomic dataset further analysis was performed to predict kinases that are active 
during enterovirus infection. The authors then pick the mTORC1 signaling pathways for more in 
depth description of its possible regulation during infection and provide limited follow up studies, 
which do not lead to conclusive biological insights in enterovirus infection. 

 

My main concerns regarding the current version of the manuscript are as follows: While the 
manuscript describes an extensive dataset that seems to be of high quality, the manuscript is mainly 
focused on describing the dataset rather than deriving biological insights into enterovirus infection 
and identifying novel host factors/signaling pathways relevant for infection. The majority of the 
paragraphs are highly descriptive, but the observations are not really linked back to the biological 
question. The authors derive some hypotheses from the dataset, but they either do not provide any 
functional follow up study to validate the hypothesis and the follow up studies they performed do 
not provide conclusive results. 

 

Some concerns/questions that I have regarding the proteomic datasets: 

Currently the authors analyze the proteome and phosphoproteome datasets separately. It would we 
important to determine whether observed changes in phosphorylation are in fact changing because 
of different levels of phosphorylation or because of protein abundance changes. This is especially 
important at later stages of infection where more changes on the proteome level are observed. 

Some of the earlier time points for the proteome analysis do not cluster at all with the replicates. Is 
this a reason why there are little significant protein abundance changes observed at earlier 
timepoints of infection? Were the proteome and phosphoproteome datasets generated from the 
same samples? 

 

In summary, the current version of the manuscript lacks novel biological insights into CVBB3 
infection and revisions are necessary to (1) put results in a biological context rather than just 
providing a detailed description, (2) provide additional functional follow up studies demonstrating 
the validity of the datasets, and (3) address the specific concerns listed below. 

 

Specific concerns: 

 

Page 4, Introduction 

The authors should provide a few sentence summary of the major findings of their study at the end 
of the introduction. What was the specific goal/motivation of the study? What was the motivation to 
use CVB3? 

 

Page 5, Results, Analysis of the proteome of infected cells 

• What is the biological motivation to use CVB3 in the study? Why was an MOI of 10 used and what 
was the infection rate obtained with this MOI? How reproducible was the infection? 

• Reproducibility of the infection seem concerning, since the clustering of the overall proteome data 
is variable and shows that replicates of earlier timepoint of the infection do not cluster at all with 
their respective biological replicates. 

• How were the clusters defined in Figure 2a (no dendogram provided)? 

• Sections of this paragraphs sound just like a description of the method and could be shortened. 

• I would suggest to restructure the manuscript. The flow of the manuscript feels interrupted, since 
the overall proteome dataset is descried in the first paragraph, but then not followed by the analysis 
of the proteome changes in the course of infection. 

 

Page 6, Results, Analysis of the phosphoproteome of infected cells 

• See above, sections of this paragraphs sound just like a description of the method and could be 
shortened. 

• How were the clusters defined in Figure 2b (no dendogram provided)? 

• It seems surprising to me that 85% of the phosphoproteome is changing in the course of infection. 
How does this compare to other phosphoproteomic datasets generated in the course of viral 
infection? How was the data normalized? Often in proteomic datasets normalization strategies are 
employed which assume that the majority of the sites are not changing. How did the authors 
address this in their data analysis? More details need to be provided in the method section? 

• See above, the manuscript feels interrupted, since the description of the phosphoproteomic 
dataset is not followed by the analysis of the phosphorylation changes. 

 

Page 6, Results, Regulation of viral protein levels during CVB3 infection 

• This paragraph is purely descriptive and out of the context in this part of the manuscript. 

• I would suggest to link it ack to CVB3 biology, otherwise I would remove it completely. 

 

Page 7, Results, Changes in host protein levels during CVB3 infection 

• I think for a better reading of the manuscript the authors should consider to restructure the result 
section and move this section after describing the proteome dataset. 

• See above, it is concerning to me that replicates of some timepoints do not cluster at all and look 
very different in the heatmap. What is the reason for this? Was there a quality control for the 
reproducibility of infection? 

• Paragraph misses more biology, what are these proteins that escape virus-induced translational 
shutdown? 

• It would also be highly interesting if the authors could perform an integrative analysis of their data 
with the CVB3 host-pathogen interaction network that was recently published in Nature 
Microbiology. 

 

Page 8, Results, Regulation of protein phosphorylation levels during CVB3 infection 

• I think for a better reading of the manuscript the authors should consider to restructure the result 
section and move this section after describing the phosphoproteome dataset. 

• At the end of the section, changes in phosphoproteome and total proteome are compared, I am 
wondering what the fraction of the phosphoproteome changes can in fact be explained by protein 
abundance changes. What is the fraction of phosphosites that change while there is no change 
observed on the protein level? 

 

Page 8, Results, Identification of kinases that are activated by CVB3 infection 

It is stated in the text that three findings of this analysis are of particular interest, such as 
overrepresentation of ATM/ATR motifs. Why are these findings of particular of particular interest? 
What does this mean for the infection process? What is the role of ATM/ATR or c PKC and GRKs in 
CVB3 infection? 

 

 

Page 9, Results, Phosphorylation of host factors involved in formation of viral replication organelles 

The authors describe in this section the phosphorylation of know proteins that might play a role in 
replication organelle formation and suggest that the specific sites might be relevant for the organelle 
formation, but again this is only descriptive and no biological follow up is provided. What is the 
possible impact of knowing the phosphorylation sites of these specific factors? Does mutating these 
sides lead to a reduction in viral replication? 

 

Page 11, Results, Signaling pathways affected during CVB3 infection 

More details need to be provided how the pathway analysis was performed. What are the 
predefined pathways known to be affected by CVB3? Where are they taken from? Was the 

proteome and phosphoproteome data just mapped onto pathways or was an enrichment analysis 
performed? Which input data was provided in for the pathway analysis, quantitative data across all 
time points? 

 

Page 11, Results, Inactivation of mTORC1 signaling during CVB3 infection 

Figure 3 associated with this section is very difficult to read: 

I would suggest now to use the blue background, which reduces the contrast in the figure. +/- 
symbols are so small that they are hard to see. Also it is not clear what the different colored arrows 
and lines mean. 

Why was a higher MOI of 50 used for validating the phosphoproteomic data by Western Blot, which 
itself was generated at an MOI of 10. 

 

Page 14, Results, Activation of the mTORC1-regulated protein transcription factor EB 

How was the knockout of TFEB performed? There is no information provided in the method section. 
Additionally, validation of the ko is missing. 

What is the explanation that 8hpi a significant decrease in virus titers is observed upon ko of TFEB, 
however there is no significant difference 10hpi? Does this makes sense? 

 

Page 18, Experimental procedures 

As mentioned in different comments above, a more detailed description of some of the methods is 
missing and should be provided.