Kaposi’s sarcoma herpesvirus viral FLICE inhibitory protein modulates A20 deubiquitinase activity
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KSHV viral FLICE inhibitory protein (vFLIP) is a potent activator of NF-κB signalling and an inhibitor of apoptosis and autophagy. Inhibition of vFLIP function and NF-κB signalling promotes lytic reactivation. Here we provide evidence for a novel function of vFLIP through inhibition of the deubiquitinating (DUB) activity of the negative regulator, A20. We demonstrate direct interaction of vFLIP with Itch and A20 and provide evidence for subsequent loss of A20 DUB activity. Our results provide further insight into the function of vFLIP in the regulation of NF-κB signalling.
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Thank you for addressing the remaining reviewers comments.
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Comments to Author
The authors have satisfactorily addressed all my concerns.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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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Thank you for responding the reviewer comments. Unfortunately these responses have not been sufficient to address their concerns, especially when it comes to scientific rigor and accurate reporting of the data. Beyond the scientific questions that remain, I am concerned about the difference in the reporting of figure 1b between this manuscript and the BioRxiv preprint. It isn't acceptable to change the methodology description (+RTA in lane 1 as described in the preprint) of this figure because it is believed that +/- RTA makes no difference to the outcome. If that is how the figure was obtained then the details need to be included, or new data obtained.
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Comments to Author
The decision made by the authors to be less definitive regarding the association between vFLIP and A20 in this revised version significantly reduces the impact of the paper. A more defined mechanism of vFLIP mediated inhibition of the A20 ubiquitinase in the absence of RTA is needed based on data that illustrates the presence or absence of a direct interaction with A20. Although the authors state that A20 was purified under denaturing conditions (further details should be provided in the manuscript), an SDS PAGE gel of purified A20 would straightforwardly address whether ITCH or other components of the editing complex were likely present and was highlighted in my responses to the original version of the paper. Furthermore, the authors can overexpress both FLAG-A20 and vFLIP so have the opportunity to …
Comments to Author
The decision made by the authors to be less definitive regarding the association between vFLIP and A20 in this revised version significantly reduces the impact of the paper. A more defined mechanism of vFLIP mediated inhibition of the A20 ubiquitinase in the absence of RTA is needed based on data that illustrates the presence or absence of a direct interaction with A20. Although the authors state that A20 was purified under denaturing conditions (further details should be provided in the manuscript), an SDS PAGE gel of purified A20 would straightforwardly address whether ITCH or other components of the editing complex were likely present and was highlighted in my responses to the original version of the paper. Furthermore, the authors can overexpress both FLAG-A20 and vFLIP so have the opportunity to perform an in-vitro pull down at the very least. My remaining queries have all been addressed.
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?
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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Comments to Author
In this paper, the authors report on the effect of KSHV vFLIP on A20 inhibition. In the initial manuscript, there were some questions about the methodological rigour of the data explaining the binding of vFLIP to A20/ITCH. And these concerns remain unresolved in the revised manuscript. The first concern is that the authors explained that the non-vFLIP-MYC band observed in lanes 1 and 3 of the IP sample in Fig. 1a was LC. If it was the LC of the antibody used for IP, an equal amount of LC should have been detected in both lanes. In the revised manuscript, the authors explained that "the amount of LC added in each sample was different". In addition, they write that the experiment was conducted three times. This mean they made the same mistake all three experiments? The authors need to present new data …
Comments to Author
In this paper, the authors report on the effect of KSHV vFLIP on A20 inhibition. In the initial manuscript, there were some questions about the methodological rigour of the data explaining the binding of vFLIP to A20/ITCH. And these concerns remain unresolved in the revised manuscript. The first concern is that the authors explained that the non-vFLIP-MYC band observed in lanes 1 and 3 of the IP sample in Fig. 1a was LC. If it was the LC of the antibody used for IP, an equal amount of LC should have been detected in both lanes. In the revised manuscript, the authors explained that "the amount of LC added in each sample was different". In addition, they write that the experiment was conducted three times. This mean they made the same mistake all three experiments? The authors need to present new data based on carefully conducted experiments with more sophisticated techniques. It should not be difficult to reacquire the data. The second concern is that the expression level of A20 in Input in Fig.1a is extremely low only in lane 3. The authors explained that overexpressed ITCH stabilizes A20. This explanation makes sense. However, IP experiment including such differences in expression levels cannot explain coprecipitation of A20 by ITCH pull-down. Thus, Figure 1a shows only the interaction between ITCH and vFLIP-MYC. On the other hand, Figure 1b shows only the interaction between FLAG-A20 and vFLIP-MYC. The question remains whether vFLIP interacts with the A20/ITCH complex or only with A20. If the authors wish to focus their report on interactions between vFLIP and A20, the manuscript needs to be substantially rewritten. On the other hand, if the author refers to the interaction of vFLIP with the complex, additional data is required. For example, they can perform pull down assay of endogenous A20 and endogenous ITCH by vFLIP-MYC. A final concern is that the data in Fig1b are clipped from a preprint published by the authors themselves in bioRxiv. It is not an issue that the same image as the preprint is used in this manuscript. However, in the manuscript, the description of lane 1 as RTA+ and lane 2 as RTA- in the preprint is ommited. Even if the presence or absence of RTA did not affect the results, it remains a concern to explain the specific coprecipitation of vFLIP-MYC by FLAG-A20 IP using samples with different RTA conditions as well as with or without FLAG-A20. The authors should re-obtain the same results. It should not be difficult to reacquire the data.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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?
Yes: There are unaddressed concerns about data representation of Figure 1B that have not been addressed in this revision.
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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Please can you address the questions and concerns of both reviewers who have both raised issues with the blot data presented and their interpretation.
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Comments to Author
The manuscript focuses on the modulation of the A20/ITCH ubiquitin editing complex by the Kaposi's sarcoma associated herpes virus encoded protein vFLIP. The paper is well written and logically organised. The methods section is sufficiently detailed although it is unclear how many replicates were generated for each of the assays featured. Despite the data being indicative of vFLIP preventing downregulation of the canonical NF-KappaB pathway through attenuation of the deubiquitinase activity of A20, which is of interest to the KSHV field and more widely, some controls are missing and their needs to be greater clarity regarding the mechanism. Of particular importance is ascertaining whether the results observed derive from a direct interaction between vFLIP and A20 and/or the requirement for ITCH. My …
Comments to Author
The manuscript focuses on the modulation of the A20/ITCH ubiquitin editing complex by the Kaposi's sarcoma associated herpes virus encoded protein vFLIP. The paper is well written and logically organised. The methods section is sufficiently detailed although it is unclear how many replicates were generated for each of the assays featured. Despite the data being indicative of vFLIP preventing downregulation of the canonical NF-KappaB pathway through attenuation of the deubiquitinase activity of A20, which is of interest to the KSHV field and more widely, some controls are missing and their needs to be greater clarity regarding the mechanism. Of particular importance is ascertaining whether the results observed derive from a direct interaction between vFLIP and A20 and/or the requirement for ITCH. My specific points are below. Major 1. In Figure 1A, the authors show evidence of ITCH/vFLIP co-elution in a complex that also involves endogenous A20 using immunoblotting. From the input lanes, however, only a small proportion of A20 appears to participate in complex formation and the authors have previously reported that vFLIP interacts with ITCH. The possibility that vFLIP could be interacting exclusively with ITCH cannot be excluded based on the data provided. Although Figure 1B shows clear co-elution of vFLIP with immunoprecipitated FLAG-A20, vFLIP could nonetheless be associating with endogenous ITCH. To address this, the FLAG-A20 immunoprecipitates should also be probed using an anti-ITCH antibody and if ITCH is detected, the experiments repeated using a cell line null for ITCH or where ITCH has been knocked down using shRNA. There should also be characterisation of the components of the A20/ITCH immunoprecipitates using mass spectrometry to establish which other elements of the A20/ITCH ubiquitin editing complex (such as RNF11 and TAXBP1) are present. 2. The A20 used in the K63-linked deubiquitinase assay shown in Figure 2 could have co-purified with endogenous ITCH or other components of the ubiquitin editing complex given the use of human 293T cells for expression. To confirm that the reduction in A20 deubiquitinase activity in the presence of vFLIP results from a direct interaction or one mediated by K63 linked ubiquitin alone, an SDS page gel of the purified protein should be provided together with an analysis using mass spectrometry to identify other components if present at significant levels. 3. Control data needs to be provided for each immunoblot experiment where re-probing for GAPDH or another "housekeeping" gene is performed to illustrate even protein loading. 4. The authors need to indicate the number of replicates for each experiment either in the methods section or in the appropriate figure legends. 5. The authors should provide an explanation for why the zero time point in the vFLIP + lanes of Figure 2 contains moderate levels of ubiquitinated RIPK1 if my interpretation is correct. Minor In Figure 2A, the concentration of vFLIP should be micromolar based on the methods section (not micrograms)
Please rate the manuscript for methodological rigour
Satisfactory
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
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data 1) The immunoprecipitation data in Figure 1A is insufficient to determine whether FLAG-ITCH is associated with A20 together with vFLIP. The amount of endogenous A20 in lane 3 of Input in Figure 1A appears significantly lower than lanes 1 and 2. The amount of Input will affect the immunoprecipitation results. I mean that similar results would be obtained if A20 was non-specifically bound to the agarose beads. Thus, Figure 1A successfully shows the interaction between ITCH and vFLIP, but does not prove the coimmunoprecipitation of A20. Results of immunoprecipitation experiments using system with no problem expressing endogenous A20 are required. 2) It is unclear what the authors wanted to show with Figure1B. Figure 1B does not …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data 1) The immunoprecipitation data in Figure 1A is insufficient to determine whether FLAG-ITCH is associated with A20 together with vFLIP. The amount of endogenous A20 in lane 3 of Input in Figure 1A appears significantly lower than lanes 1 and 2. The amount of Input will affect the immunoprecipitation results. I mean that similar results would be obtained if A20 was non-specifically bound to the agarose beads. Thus, Figure 1A successfully shows the interaction between ITCH and vFLIP, but does not prove the coimmunoprecipitation of A20. Results of immunoprecipitation experiments using system with no problem expressing endogenous A20 are required. 2) It is unclear what the authors wanted to show with Figure1B. Figure 1B does not specifically demonstrate RTA independency. The results obtained in Figure 1B may be included in those obtained in Figure 1A ((RTA-independent) interaction between vFLIP and A20/Itch complex). Figure 1B may not be necessary if the authors have created a modified Figure 1A. 3) The authors' preprint that are closely related to this research are presented in biorxiv. Figure 1B in this paper appears to derived from the same source as Figure 1B in that preprint. If so, lane 1 in Figure 1B would be with RTA and lane 2 would be without RTA, but it was not explained in this paper. If the authors publish Figure 1B, they should reacquire the same data under the conditions described in this paper. 2. Presentation of results The presentation of results is clear. 3. How the style and organization of the paper communicates and represents key findings The style and organization of the paper are suitable for presenting important findings. 4. Literature analysis or discussion The literature used in this manuscript provides sufficient coverage of the background and key findings. But the discussion is not enough. Based on the obtained results, please discuss the intermolecular interactions of vFLIP, ITCH and A20. 5. Any other relevant comments 1) If the LC is due to anti-M2 Flag antibodies, the LC band should be visible at the same intensity in all lanes. Why is the LC band not visible in lane 2 of IP:ITCH in Figure 1A? A reasonable explanation is required. 2) In the legend of Figure 1B (line 114), the authors say "Itch was immunoprecipitated with Flag antibody," but it was FLAG-A20 that was immunoprecipitated in Figure 1B. 3) In Figure 2, the authors clearly demonstrated that vFLIP inhibits the DUB activity of A20 without ITCH. Does A20 directly associated with vFLIP under the in vitro condition? Coprecipitation in in vitro reconstituted systems may provide more direct evidence of interaction than coprecipitation in cell lysates. Clarifying the intermolecular relationship between vFLIP and A20 by immunoprecipitation in vitro should improve the quality of this paper.
Please rate the manuscript for methodological rigour
Poor
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
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