Review 3: "Protein arginylation is regulated during SARS-CoV-2 infection"

RR:C19 Evidence Scale rating by reviewer:

Not informative. The flaws in the data and methods in this study are sufficiently serious that they do not substantially justify the claims made. It is not possible to say whether the results and conclusions would match that of the hypothetical ideal study. The study should not be considered as evidence by decision-makers.

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Review:

I believe that the paper entitled “Protein arginylation is regulated during SARS-CoV-2 infection” needs significant corrections and additional experiments before publication.

1. Page 9, last sentence: I agree with a similar expression pattern of potential arginylated-proteins, but it is unclear why the author chose the various cell types.  For example, the Vero E6 cell line is from monkey kidney tissue, and Caco-2 is from the human colon.  The relationship between these cell lines or the reasoning behind their selection should be explained in the manuscript.  If the author chose these based on the available datasets, please explain this limitation in the manuscript.  Also, the last words ‘Green Monkey and Human’ should be changed to ‘human (Green) and monkey (Yellow).

2. Figure 2 micrograph B:  Please replace the western blots provided.  The current blots shown are of low quality and not fully convincing.  For instance, the 24 hour time point in the Calu-3 blot seems significantly down-regulated even compared with the GAPDH level.  Analysis with software such as ImageJ would be helpful to see the actual pattern of ATE1 expression, but given the poor quality of these blots, accurate results may be hard to obtain.

3. Page 11, last paragraph:  According to the argument, Calu-3 cells are less susceptible to infection, but what evidence supports this?  For Figure S1, micrographs A and B,  please add size markers on western blot results.  Please ensure the minimal size marker, at least >140kDa, for showing poly-ubiquitination. 

4. The authors mentioned that SARS-Cov-2 infection induces ER stress, but BiP, a representative marker/indicator of ER stress, was not upregulated as expected.  Furthermore, the authors’ claim that SARS-Cov-2 infection upregulated arginylated BiP is not supported by the data shown in Figure 4. Western blots showing arginylated ER chaperones (BiP, CALR, and PDI) need an ATE1 knockdown control proving that the antibodies detect the chaperones’ arginylated form.

5. Page 19, Figure 6:  The western blot labeling is missing.  This problem is repeated in Figure 7.  In addition, the poor quality of the western blots and contrast manipulation make interpretation difficult when scanning for quantification.   Visually there seems to be no difference between the infected 24h sample and the infected t.acid 24h sample in terms of ATE1 expression, but the quantification shows a drastic decrease.  Why does the ATE1  quantification show a drastic increase in ATE1 expression in the tannic acid control compared with the normal untreated control?  Looking at the blot, the GAPDH for the untreated control is magnitudes less than the tannic acid control, and the difference is greater than the difference in ATE1 bands for these samples.  The expected quantification result would be a similar ATE1 expression between the untreated and tannic acid controls.  This calls into question the quantification methods being used to quantified these blots and the conclusion being drawn.  There are similar issues with the other blots. 

6. There is no direct evidence that both Tannic acid and Merborin are specific inhibitors of ATE1.  Therefore, in addition to these inhibitors, ATE1 RNA interference using either siRNA or shRNA should be used to determine ATE1’s specific influence on viral load.