Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication

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1 Manuscript EMBO Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication Tsai-Ling Liao, Chung-Yi Wu, Wen-Chi Su, King-Song Jeng and Michael Lai Corresponding author: Michael Lai, University of Southern California Review timeline: Submission date: 12 May 2010 Editorial Decision: 11 June 2010 Revision received: 05 August 2010 Editorial Decision: 11 August 2010 Revision received: 25 August 2010 Additional Correspondence: 30 August 2010 Additional Correspondence: 01 September 2010 Additional Correspondence: 02 September 2010 Accepted: 14 September 2010 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 11 June 2010 Thank you for submitting your manuscript for consideration by the EMBO Journal. It has now been seen by three referees whose comments are enclosed. As you will see, all three referees express interest in your study, but while referee 1 has only minor comments, referees 3 and particularly 2 raise a number of more serious concerns that would need to be addressed before we could consider publication of your manuscript in the EMBO Journal. I would in particular like to draw your attention to the comments of referee 2, who finds that further evidence that NP is indeed ubiquitinated on K184 would be essential. Moreover, he/she argues that, if K184 is indeed the ubiquitination site, this might be predicted to disrupt RNA binding - something that could and should be tested without too much difficulty. Addressing these criticisms would be critical for possible acceptance of your manuscript. Given the overall positive recommendations, I would therefore like to invite you to submit a revised version of the manuscript, addressing all the comments of all three reviewers. I should add that it is EMBO Journal policy to allow only a single round of revision. Acceptance of your manuscript will thus depend on the completeness of your responses included in the next, final version of the manuscript. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process initiative, please visit our website: We generally allow three months as a standard revision time, and as a matter of policy, we do not consider any competing manuscripts published during this period as negatively impacting on the European Molecular Biology Organization 1

2 conceptual advance presented by your study. However, we request that you contact the editor as soon as possible upon publication of any related work, to discuss how to proceed. Should you foresee a problem in meeting this three-month deadline, please let us know in advance and we may be able to grant an extension. Thank you for the opportunity to consider your work for publication. I look forward to your revision. Yours sincerely, Editor The EMBO Journal REFEREE REPORTS Referee #1 (Remarks to the Author): Liao et al identified a cellular deubiquitinating enzyme, USP11, in a screen for affects on influenza virus replication and further showed that USP11 negatively regulated viral genomic RNA replication by a mechanism that requires the catalytic activity of USP11. They went on to show that USP11 interacts with the viral replication proteins PB2, PA and NP and reverses the monoubiquitination of NP in vivo. They also identified the site of monoubiquitination in NP and showed that changing this K to R inhibited viral replication. Together the results indicate that USP11 can regulate influenza virus replication through deubiquitination of NP. This study is novel in identifying a functional role of USP11 in influenza replication and in identifying NP regulation through monoubiquitination. The data is thorough and convincing. My only minor comment is as follows: On page 8, the authors mention screening sirna for 53 human DUBs for affects on influenza replication. It would be useful for others in the field to know which DUBs were screened and whether any others affected viral replication. Perhaps a table or supplemental data could be included to summarize these results. Referee #2 (Remarks to the Author): These investigators present results concerning the ubiquitination of the influenza virus NP protein and the role of this ubiquitination during virus infection. They show that USP11, a nuclear ubiquitin deconjugating enzyme, inhibits influenza virus infection by inhibiting viral genome (vrna) replication. Although the effects of shrnai knockdown and USP11 overexpression are not dramatic (2-5-fold), the results support the authors' conclusion that USP11 inhibits vrna synthesis during infection. In addition, the transient transfection experiments demonstrate that NP can be conjugated to ubiquitin, that the conjugation site is at NP amino acid 184, and that USP11 can remove the ubiquitin from NP. Unfortunately, however, the ensuing experiments are unconvincing, and crucial experiments are not carried out. Some of the problems are: 1. The authors never establish that NP is ubiquitinated at amino acid 184 during virus infection. The mini-replicon assays are also transient transfection experiments (page 15, bottom paragraph), and do not address events in virus-infected cells. In addition, the authors presume that the defect seen with the K184R mutant is due to the lack of ubiquitination, whereas the larger defect seen with the K273R mutant is due to some other issue. There is no evidence for this conclusion. It is also crucial to know whether USP11 inhibits the activity of the wild-type protein and all the KR mutant proteins other than the K184R mutant protein, thereby demonstrating that ubiquitination of NP enhances viral RNA replication in this assay. 2. The only experiments in infected cells use a single virus mutant, a virus expressing a K184R mutation, and the authors assume without any direct evidence that NP is ubiquitinated and that K184 is the site of ubiquitination in infected cells. In fact, it should not be difficult to establish whether this is the case. The putative defects in viral RNA replication in cells infected by the K184R European Molecular Biology Organization 2

3 mutant virus could be due to effects other than the lack of ubiquitination. The virus growth curves shown in Figures 6E and 6F are not acceptable. It is essential to show the actual amounts of virus produced at each time point by the wild-type and K184R mutant virus. In fact, the K184R mutant replicates only 2-fold less than the wild-type virus in A549 cells (Figure 6F), and the reduction in vrna synthesis is also quite small (2-fold). These results are not convincing. 3. Amino acid K184R is located in the RNA-binding groove of the NP protein, and it should be extremely easy to determine how ubiquitination of this amino acid and a K-to-R substitution at this position affects the RNA-binding activity of the NP protein. In the absence of such functional results, the authors cannot make any clearcut statement about the role of K184 ubiquitination. Referee #3 (Remarks to the Author): This study describes a novel interaction between influenza NP and a cellular deubiquitinase, USP11. Using an RNAi-based screen the authors identify USP11 as an inhibitor of influenza virus replication and more specifically vrna and crna synthesis. The deubiquitinase activity is required for the inhibitory activity. USP11 is shown to interact with NP, PB2 and PA and the monoubiquitinated NP is a substrate for USP11. The site of ubiquitination was identified as lysine 184 and a recombinant virus with a K184R mutation in NP is shown to be attenuated for growth in cell culture with decreases in vrna and crna synthesis. Overall the authors propose that NP ubiquitination on K184 controls the switch between primary transcription and replication and that USP11 can inhibit this switch. This is a particularly interesting story that identifies a critical role for mono-ubiquitinated NP in regulating influenza virus genome replication and identifies a cellular protein that inhibits this activity. The experiments are all well-designed, appropriately controlled and fully support the conclusions drawn by the authors. Such results provide a significant advance to the field and further our understanding of this essential part of the influenza virus life cycle. Comments to be addressed: 1. Is USP11 an interferon-inducible protein? 2. What do the authors think is the basis for the specificity of USP11 vs. USP10? Is USP10 in the nucleus as well? 3. Figure 2A. Which of these 3 clones qualified as hits in the screen and does this correlate with the knockdown efficiency observed here? 4. Figure 3A. Why isn't a secondary effect on mrna (i.e. an increase) seen in the presence of USP11 shrna? 5. Figure 5B. It appears that the NP:USP11 interaction is not dependent on ubiquitination. Do the authors agree and can this be confirmed with the NP K184R mutant? 6. Figure 6B. Is K184 (or the equivalent) also conserved in influenza B viruses? 7. Figure 6D. Was this also tried in the context of the full vrnp? i.e. add in PA and the minigenome reporter. 8. Figures 6E and F. In E, these are very early time points to perform with a low MOI infection. Increase the timecourse to at least 48h. For both E and F, show actual PFU/ml titers so that the growth properties can be accurately assessed. 9. Page 4. Replace E3s with E3 ligases (X2) 10. Page 7-8. If ISG15 was used as a positive control, does this imply that the screen was specifically looking for inhibitors? This is unclear. 11. It's also not clear how many genes were screened. Was it only the 52 human DUB genes and was USP11 the only hit? 12. Page 9, lines 6-7. Remove the sentence starting "Similarly,...". Already said above (Fig 2B). 13. Pg 14, first line. Replace "above" with Fig 4C and 5A. 14. Lastly, there is a recent report in PNAS regarding the role of viral small RNAs in the switch from transcription to replication. In the discussion please comment on these findings. European Molecular Biology Organization 3

4 1st Revision - authors' response 05 August 2010 Referee #1 On page 8, the authors mention screening sirna for 53 human DUBs for affects on influenza replication. It would be useful for others in the field to know which DUBs were screened and whether any others affected viral replication. Perhaps a table or supplemental data could be included to summarize these results. Response: We thank referee #1 s remark. We have already added the information of DUB subset for RNAi screening as supplementary Table I. Referee #2 1. The authors never establish that NP is ubiquitinated at amino acid 184 during virus infection. The mini-replicon assays are also transient transfection experiments (page 15, bottom paragraph), and do not address events in virus-infected cells. In addition, the authors presume that the defect seen with the K184R mutant is due to the lack of ubiquitination, whereas the larger defect seen with the K273R mutant is due to some other issue. There is no evidence for this conclusion. It is also crucial to know whether USP11 inhibits the activity of the wild-type protein and all the KR mutant proteins other than the K184R mutant protein, thereby demonstrating that ubiquitination of NP enhances viral RNA replication in this assay. Response: We thank referee #2 for careful reading and useful comments. In the revised manuscript, we performed an experiment to proof NP is ubiquitinated at K184 during virus infection. USP11 knockdown cells were transfected with a plasmid that can express Myc-Ub, then infected with NP- K184R mutant or wild-type virus respectively. Cell lysates were used for immunoprecipitation with anti-ub antibody. The immunoblot result showed that NP-Ub form can be detected in wild-type, but not in NP-K184R mutant virus (Fig. 6H). We also performed an experiment to dissect the mechanism of inhibition of viral RNA replication by USP11 (Fig. 6I). USP11 knockdown or control cells were infected with WT or K184R mutant virus at MOI of 5, and total cellular RNA was used for primer extension analysis at 8 hours postinfection. The result showed that when viral NP protein was defective in ubiquitination (K184R), there was no dramatic difference in viral RNA synthesis between USP11 knockdown and control cells (Fig. 6I lanes 4, 5 and 6), suggesting that USP11 inhibits viral RNA replication through deubiquitinating NP. Once NP cannot be ubiquitinated, such as in K184R mutant virus, the effect of cellular USP11 inhibition was diminished. Therefore, we conclude that USP11 inhibits viral RNA replication through deubiquitinating NP. In the case of K273, the published result demonstrated that it is a crucial amino acid essential for interaction of NP with the polymerase subunits (PB1 and PB2) (Biswas et al, 1998; Li et al, 2009b). 2. The only experiments in infected cells use a single virus mutant, a virus expressing a K184R mutation, and the authors assume without any direct evidence that NP is ubiquitinated and that K184 is the site of ubiquitination in infected cells. In fact, it should not be difficult to establish whether this is the case. The putative defects in viral RNA replication in cells infected by the K184R mutant virus could be due to effects other than the lack of ubiquitination. The virus growth curves shown in Figures 6E and 6F are not acceptable. It is essential to show the actual amounts of virus produced at each time point by the wild-type and K184R mutant virus. In fact, the K184R mutant replicates only 2-fold less than the wild-type virus in A549 cells (Figure 6F), and the reduction in vrna synthesis is also quite small (2-fold). These results are not convincing. Response: We have already revised the virus growth curve presentation to show the actual viral titer (Fig. 6F and 6G). We also showed the viral titers at longer time points post-infection (Fig. 6G). At 24 hours post-infection, the difference in virus titer was increased to about 10-fold. At 10 hours post-infection, the difference in vrna synthesis was about 4 fold. But at the time-points, the mrna amount also decreased, probably as a secondary effect of reduction of vrna as crna. 3. Amino acid K184R is located in the RNA-binding groove of the NP protein, and it should be extremely easy to determine how ubiquitination of this amino acid and a K-to-R substitution at this position affects the RNA-binding activity of the NP protein. In the absence of such functional results, the authors cannot make any clear cut statement about the role of K184 ubiquitination. European Molecular Biology Organization 4

5 Response: We have performed an in vivo RIP assay to proof that ubiquitination at K184 can alter the RNA-binding activity of NP (Fig. 6E). Actually, because the specific ubiquitination E3 ligase for NP still unknown, we can t perform in vitro assay (such as UV cross-linkage or gel shift) to study RNA binding activity of NP-K184R mutant. This in vivo RIP assay showed that NP-K184R mutant vrna binding activity was reduced to approximately 20% of that of the wild-type NP (lane 5). Furthermore, with ubiquitin, the vrna binding activity of the wild-type NP was increased by about 2.5 fold as compared with the non-ubiquitinated state (lane 4). Referee #3 Response: We thank referee #3 for carefully reading. The response for each question is as followed: 1. Is USP11 an interferon-inducible protein? Response: No. 2. What do the authors think is the basis for the specificity of USP11 vs. USP10? Is USP10 in the nucleus as well? Response: Based on our result (Fig. 5D), although USP11 and USP10 both can interact with NP, only USP11 can cleave single ubiquitin from NP-Ub specifically. In addition, USP10 is not a nuclear protein; it is located in both the cytoplasm and nucleus. 3. Figure 2A. Which of these 3 clones qualified as hits in the screen and does this correlate with the knockdown efficiency observed here? Response: The RNAi screening result showed that the cell viability ratio value is 0.32, 0.35, 0.34, respectively for these 3 clones, (Please see supplementary Table I). All these three fit the criterion for determining hits of screening and also were confirmed by immunoblot result (Fig. 2A). The immunoblot data showed that clone 5 has the best knockdown efficiency. Therefore, clone 5 was used for further studies. 4. Figure 3A. Why isn't a secondary effect on mrna (i.e. an increase) seen in the presence of USP11 shrna? Response: In Fig. 3A, USP11 knockdown cells were used to check cellular USP11 function during influenza virus infection. The result showed that only viral RNA replication was inhibited and that there was no secondary effect on mrna synthesis. In Fig. 3B, USP11-overexpressing cells were used and showed secondary effects on mrna. We hypothesize that when cellular USP11 was overexpressed, the interaction between USP11 and NP or, between PB2 and PA is increased; therefore, secondary effect is easier to detect. 5. Figure 5B. It appears that the NP:USP11 interaction is not dependent on ubiquitination. Do the authors agree and can this be confirmed with the NP K184R mutant? Response: We cannot make this conclusion based on Fig. 5B alone. The result in Fig. 5B just indicated that both USP11 and USP11 mutant can interact with NP. Actually, we performed the experiment as you suggested, namely, we checked the interaction between USP11 and NP-K184R mutant. The immunoprecipitation result showed that NP-K184R can interact with USP11, but the amount of immunoprecipitated protein was lower than wild-type (data not shown). 6. Figure 6B. Is K184 (or the equivalent) also conserved in influenza B viruses? Response: Yes. We have revised Fig. 6B and added influenza B virus sequence. 7. Figure 6D. Was this also tried in the context of the full vrnp? i.e. add in PA and the minigenome reporter. Response: No, because the literature showed that NP interacted with PB1 and PB2, but not PA European Molecular Biology Organization 5

6 (Biswas et al, 1998). We only focused on the interaction between NP and PB1 or PB2. 8. Figures 6E and F. In E, these are very early time points to perform with a low MOI infection. Increase the time course to at least 48h. For both E and F, show actual PFU/ml titers so that the growth properties can be accurately assessed. Response: We have already revised the virus growth curve presentation to show the actual virus titer (Fig. 6F and 6G). We also showed the result at longer time point post-infection (Fig. 6G). At 24 hours post-infection, the difference in virus titer was increased to about 10-fold. 9. Page 4. Replace E3s with E3 ligases (X2) Response: Already revised. 10. Page 7-8. If ISG15 was used as a positive control, does this imply that the screen was specifically looking for inhibitors? This is unclear. Response: Yes, in our RNAi screening condition (infected FluA with MOI at 1), the hit genes are inhibitor. The cell viability ratio of candidate clones were all below the cut-off value. 11. It's also not clear how many genes were screened. Was it only the 52 human DUB genes and was USP11 the only hit? Response: There are 262 clones in the TRC RNAi DUB library, which includes 52 human DUB genes for our RNAi screening. USP11 gave the most clear cut result. We also picked up another 2 candidate genes. But one had been ruled out and the other is still being characterized. 12. Page 9, lines 6-7. Remove the sentence starting "Similarly,...". Already said above (Fig 2B). Response: Already revised. 13. Pg 14, first line. Replace "above" with Fig 4C and 5A. Response: Already revised. 14. Lastly, there is a recent report in PNAS regarding the role of viral small RNAs in the switch from transcription to replication. In the discussion please comment on these findings. Response: This report showed that influenza A virus-derived small viral RNAs (svrnas) can trigger the viral switch from transcription to replication through interactions with the viral polymerase machinery. Our result (Fig. 6D) showed that, there were no significant differences between the interactions of NP-K184R and wild-type NP with PB2 or PB1, which are subunits of viral polymerase. In addition, our study also showed that ubiquitination of NP on K184 can alter the interactions of NP with RNA (Fig. 6E), thus stabilizing crna, and thereby increase RNA replication efficiency (Fig 7A), but not transcription. It is possible that multiple factors regulate viral RNA replication and transcription. 2nd Editorial Decision 11 August 2010 Many thanks for submitting the revised version of your manuscript EMBOJ It has now been seen again by referee 2, whose comments are enclosed below. As you will see, he/she is unfortunately not satisfied that you have adequately addressed his/her concerns from the previous round of review, and consequently does not recommend publication of your study. The major criticism continues to be that you have not adequately demonstrated that it is the lack of ubiquitination in the K184R mutant, rather than any other potential effect of this mutation, that is responsible for the observed phenotype. European Molecular Biology Organization 6

7 Given the positive recommendations of referees 1 and 3 in the initial round of review, and since the criticisms of referee 2 are primarily technical in nature, we would be willing to offer you the chance to undertake a second round of revision to address these remaining concerns of referee 2. Most critical here would be extending the mini-replicon analysis shown in Figure 6C, to demonstrate that knock-down of USP11 in this assay reduces replication to a degree similar to that of the K184R mutation, while having no effect on the K184R mutant construct. The experiment you show in Figure 6I, which does address this point in another way, would also need to be modified and repeated so as to provide quantitative data. I would stress that potential acceptance of your manuscript would be absolutely contingent upon your fully addressing all of the concerns raised by referee 2 in his/her report. Should you be unwilling or unable to conduct these additional experiments, I would suggest that you instead take your manuscript elsewhere at this point (in which case, please let me know so we can withdraw your manuscript from the system). If you would like to discuss this further, please don't hesitate to get in touch. I do hope that you will be able to address these remaining concerns, and that we can still achieve a positive outcome here! With best wishes, Editor The EMBO Journal REFEREE REPORT Referee #2 (Remarks to the Author): The authors have not appropriately addressed my criticisms of their original manuscript. As a consequence, the manuscript remains seriously flawed: 1. The authors have failed to address my criticism of their interpretation of their mini-replicon results. I requested that they determine "whether USP11 inhibits the activity of the wild-type protein and all the KR mutant proteins other than the K184R mutant protein, thereby demonstrating that ubiquitination of NP enhances viral RNA replication in this assay." The authors did not provide the requested USP11 knockdown results, so that there is no basis at all for the authors' statement on page 15, lines that the 30% reduced activity in this assay is "most likely due to failure of ubiquitination." Why did the authors not provide these straightforward results? The results shown in Figure 6I do not compensate for the failure to provide the requested results. In fact, the results of Figure 6I are not convincing. These primer extension results are certainly not quantitative, and the authors need to carry out quantitative RT-PCR to substantiate their claims of a "dramatic" difference after USP11 knockdown. A "dramatic" difference is not apparent. 2. The experiment to demonstrate that K184 is the site of NP ubiquitination in infected cells is flawed. The authors transfect a plasmid expressing myc-ub, followed by infection. Instead of taking advantage of potent myc antibody for immunoprecipitation, the authors use the much less potent Ub antibody. This makes no sense at all, and results in very light bands in the NP immunoblots (Figure 6H). Consequently, retention of any Ub by the K184R mutant would be very difficult to detect. The authors need to show the results of experiments in which: (i) the myc antibody is used for immunoprecipitation, followed by an immunoblot with NP antibody; and (ii) in which the myc antibody is used for an immunoblot after immunoprecipitation with the NP antibody. 3. The authors now show a small defect in the replication of the K184R virus relative to the wild-type virus (Figure 6G), but there is no evidence that this is due to the absence of ubiquitination of K184, rather than due to a defect caused simply by replacing R for K at this position. The authors need to show that USP11 knockdown eliminates this small difference in virus replication. European Molecular Biology Organization 7

8 2nd Revision - authors' response 25 August 2010 We thank referee #2 very much for reviewing our manuscript very carefully and giving useful comments to improve the quality of our manuscript. The response to each question is as follows: Referee #2 The authors have not appropriately addressed my criticisms of their original manuscript. As a consequence, the manuscript remains seriously flawed: 1. The authors have failed to address my criticism of their interpretation of their minireplicon results. I requested that they determine "whether USP11 inhibits the activity of the wildtype protein and all the KR mutant proteins other than the K184R mutant protein, thereby demonstrating that ubiquitination of NP enhances viral RNA replication in this assay." The authors did not provide the requested USP11 knockdown results, so that there is no basis at all for the authors' statement on page 15, lines that the 30% reduced activity in this assay is "most likely due to failure of ubiquitination." Why did the authors not provide these straightforward results? The results shown in Figure 6I do not compensate for the failure to provide the requested results. In fact, the results of Figure 6I are not convincing. These primer extension results are certainly not quantitative, and the authors need to carry out quantitative RT-PCR to substantiate their claims of a "dramatic" difference after USP11 knockdown. A "dramatic" difference is not apparent. Response: We feel apologetic about misunderstanding referee #2 s request about Fig.6C in the initial round of review. We repeated the mini-replicon experiment in USP11 knockdown cells at the reviewer s request. We also co-transfected with a plasmid that can express Ub as a control. The result of this new experiment (Fig.6C) showed that the luciferase activity of K184R was reduced to 15% of the wild type in USP11 knockdown cells. This result was even more clear-out than that shown in normal cells (30%, please see supplementary Fig. S1). In addition, when cells overexpressed Ub, the luciferase activity was increased in most of NP constructs (including WT, K113R, K229R and K325R), but not in K184R. We believe that these new data support the conclusion we previously hypothesized on page 15 that ìreduced activity in this assay is most likely due to failure of ubiquitinationî and should address the reviewer s concerns. We thank referee #2 s suggestion which made this conclusion more convincing. In order to conform to the rule of figure size limitation in the EMBO Journal and maintain the integrity of the manuscript, we remove original Fig. 6I to Fig. 7A. In addition, we had performed quantitative RT-PCR to correlate the primer extension result as done in Fig. 3 and Fig. 6G. We didn t show the qrt-pcr result in the original manuscript, because we thought that the difference in vrna amount in primer extension data was sufficiently clear and that there was size limitation for figure in the journal. In any case, the qrt-pcr data are included at the referee s request. These data are included in Fig. 7A (original Fig. 6I). 2. The experiment to demonstrate that K184 is the site of NP ubiquitination in infected cells is flawed. The authors transfect a plasmid expressing myc-ub, followed by infection. Instead of taking advantage of potent myc antibody for immunoprecipitation, the authors use the much less potent Ub antibody. This makes no sense at all, and results in very light bands in the NP immunoblots (Figure 6H). Consequently, retention of any Ub by the K184R mutant would be very difficult to detect. The authors need to show the results of experiments in which: (i) the myc antibody is used for immunoprecipitation, followed by an immunoblot with NP antibody; and (ii) in which the myc antibody is used for an immunoblot after immunoprecipitation with the NP antibody. Response: We thank referee #2 s suggestion. Actually, we had already performed this experiment in the original manuscript but didn t show this result, because we thought that direct demonstration of ubiquitination of NP using anti-ub antibody is more convincing. In the re-revised manuscript, we added the immunoprecipitation (IP) with anti-myc antibody and also anti-ubiquitin and immunoblot (IB) with anti-np antibody result in upper panel of Fig. 6H. As referee s prediction, the signal was stronger with anti-myc than with anti-ub antibody (middle panel); under this condition, there was still no signal detected in K184R mutant virus. We did not perform IP with anti-np, because there is no commercial anti-np antibody for IP available. In any case, the reverse experiment, IP with antimyc and IB with anti-np, has been done. We hope that this is satisfactory. European Molecular Biology Organization 8

9 3. The authors now show a small defect in the replication of the K184R virus relative to the wild-type virus (Figure 6G), but there is no evidence that this is due to the absence of ubiquitination of K184, rather than due to a defect caused simply by replacing R for K at this position. The authors need to show that USP11 knockdown eliminates this small difference in virus replication. Response: We agree with the referee s concern. In fact, we performed both USP11 knockdown and over-expression experiments. The results are shown in Fig. 6C, 7A, 7B and S2 using both minireplicon and virus infection approaches. In the re-revised manuscript, two new figures (Fig. 6C and Fig. 7A) were added, showing that NP-K184R was defective in RNA replication and that the ubiquitinating enzymes failed to rescue this defect. In contrast, ubiquitination enhanced RNA replication of wild type. The recombinant K184R mutant virus RNA replication result shown in Fig. 6G is consistent with this result. The most convincing data came from Fig. 7A, which included the quantitative RT-PCR result as requested by the referee. The difference in the amount of vrna between WT and K184R mutant virus was about 10 fold in USP11 knockdown cells (lane 1 vs. lane 4). We also performed a mini-replicon experiment in USP11 over-expressing cells (Supplementary Fig. S2). The luciferase activity of wild-type NP was decreased to the same level as the K184R mutant in USP11 over-expressing cells; but in cells expressing the catalytically defective USP11 mutant or control cells, the luciferase activity of the wild-type NP was significantly higher than that of the K184R mutant. The activity of K184R mutant was not affected by the over expression of USP11. The same conclusion was obtained when wild-type and K184R mutant viruses were for infection in USP11-over-expressing cells (Fig. 7B, detail mentioned in the text p.20-21). These results combined should be able to address the referee s questions. Additional Correspondence 30 August 2010 Many thanks for submitting the latest version of your manuscript. I have now had the chance to look through it and your response to the referee's remaining criticisms. I'm afraid I still have a concern regarding the mini-replicon assay. The reviewer specifically asked you to compare the effects of the various NP mutants in wt vs. USP11 knockdown cells. While you now show the data from the USP11 knockdown cells, you do not directly compare the replication rates between wt and KD. Moreover, you state in your point-by-point response that the effects of the K184R mutation are even stronger in the USP11 knockdown than in wild-type. Since you are proposing that USP11 acts only by de-ubiquitinating K184, it's not clear to me why you should see this difference. Do you have directly comparable results for wt vs. USP11 (i.e. from experiments conducted at the same time)? Is there a reason why these data are not shown? I'm sorry to insist on this, but both the referee and I were very specific that these data were important to the manuscript, and I'm somewhat concerned that you still don't show the requested experiment. I do agree that the data presented in Figure 7 are reasonably convincing on this front, but I would like to see the replicon assays shown as well. Please can you get back to me and let me know whether you have these data, or would be able to generate them rapidly; or, alternatively, whether there is a specific reason why you have not followed the referee's request. Many thanks for your cooperation here! Best wishes, Additional Correspondence 01 September 2010 We thank the editor and referees very much for very careful reading of our manuscript. The response to mini-replicon result is as follows: European Molecular Biology Organization 9

10 I'm afraid I still have a concern regarding the mini-replicon assay. The reviewer specifically asked you to compare the effects of the various NP mutants in wt vs. USP11 knockdown cells. While you now show the data from the USP11 knockdown cells, you do not directly compare the replication rates between wt and KD. Moreover, you state in your point-by-point response that the effects of the K184R mutation are even stronger in the USP11 knockdown than in wild-type. Since you are proposing that USP11 acts only by de-ubiquitinating K184, it's not clear to me why you should see this difference. Do you have directly comparable results for wt vs. USP11 (i.e. from experiments conducted at the same time)? Is there a reason why these data are not shown? I'm sorry to insist on this, but both the referee and I were very specific that these data were important to the manuscript, and I'm somewhat concerned that you still don't show the requested experiment. I do agree that the data presented in Figure 7 are reasonably convincing on this front, but I would like to see the replicon assays shown as well. 1. The mini-replicon experiment you and the referee #2 insisted on has been performed previously (now Supplementary Figure S3). The result is comparable to that of Fig. 7A. The only difference between the two experiments is that Fig. 7A was done with the WT and K184 mutant VIRUSES, whereas Fig. S3 was done with WT and mutant PLASMIDS (replicon). As you can see, the results were comparable. These experiments were done in the USP11KD and control cells, precisely as you requested. We did not present the plasmid figure (Fig. S3) in the previous version of the manuscript was because we thought that the experiment using live virus infection would be more convincing than those done with plasmid transfection. In any case, we now include this piece of data as a supplementary figure. If you feel that we should include it as a regular figure, we would be very happy to do so. It will take a few more days to complete. In fact, the issue raised by you and the reviewer has also been addressed in Fig. 6C, in which the mutant and WT replicons were transfected into USP11KD cells with or without the over-expressed ubiquitin. Thus, this is exactly the complementary approach to that of the supplementary Figure S3. It is noted that the luciferase activity of K184R mutant was not affected by over-expression of ubiquitin. In the same figure (Fig. 6C), we demonstrated that all the other NP mutants were stimulated by over-expression of ubiquitin. Thus, we believe that we have provided more experimental evidence than requested by the reviewer. However, if you feel that we need to repeat the replicon experiment for all the mutants in USP11KD vs. wild-type cells, we would be happy to perform the experiment. 2. We mentioned that the effects of K184R mutation are stronger in USP11 knockdown cells (Fig. 6C, black bars, 15% of the WT plasmid) than in wild-type cell (Fig. S1, 30% of the WT plasmid). This statement was based on the data normalized relative to the value of wild-type NP in each cell. Actually, the replication rates of K184 mutant in both cells were similar, but the replication rate of wild-type NP was higher in USP11 knockdown cells, thus affecting the relative ratio. We think that this statement is confusing and should be deleted. Additional Correspondence 02 September 2010 Many thanks for getting back to me and for your clarifications. I had actually missed the reference to Supplementary Figure 2, where you show the direct comparison I asked for - many apologies for that! Presumably the fact that replication is higher in USP11 KD vs. wt suggests that USP11 has other functions in this context beyond just deubiquitinating NP - perhaps a statement to this effect would be valuable? Given your explanations, I'm happy to publish you study: could you please just make the final few changes to the manuscript (since these should just be text changes, the easiest thing would be for you to send an updated version of the text file to us by , and we can replace it in the system). We can then go ahead and accept the manuscript. Thanks for your cooperation with this, and best wishes, European Molecular Biology Organization 10