The immediate early protein 1 of human herpesvirus 6B counteracts ATM activation in an NBS1-dependent manner

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   Mar 7, 2023

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   Manuscript number: RC-2022-01785

   Corresponding author(s): Amélie, Fradet-Turcotte, and Louis, Flamand

   Title: The immediate early protein 1 of human herpesvirus 6B counteracts ATM activation in an NBS1-dependent manner

   Our manuscript received positive and constructive comments from all three Reviewers. First, they unanimously agreed that the biology uncovered in our study is novel and of broad scientific interest, including researchers studying host-pathogen, DNA damage response and repair processes. They highlighted that the manuscript is well-written and presents clear, rigorous, and convincing data. Second, they provided constructive comments to …

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   Reply to the reviewers

   Manuscript number: RC-2022-01785

   Corresponding author(s): Amélie, Fradet-Turcotte, and Louis, Flamand

   Title: The immediate early protein 1 of human herpesvirus 6B counteracts ATM activation in an NBS1-dependent manner

   Our manuscript received positive and constructive comments from all three Reviewers. First, they unanimously agreed that the biology uncovered in our study is novel and of broad scientific interest, including researchers studying host-pathogen, DNA damage response and repair processes. They highlighted that the manuscript is well-written and presents clear, rigorous, and convincing data. Second, they provided constructive comments to strengthen our model and the biological relevance of our findings. Here, we provide an overview of our findings and a point-by-point reply explaining the revisions, additional experimentations and analyses planned to address the points raised by the referees.

   1 - Description of the planned revisions

   1.1 *The three Reviewers agreed that we convincingly show that HHV-6B IE1 binds to NBS1 and inhibits ATM activation; however, they all raised concerns about whether the IE1-dependent inhibition of ATM is required for HHV-6B replication and integration. *

   We agree with the Reviewers that the biological data validating the impact of ATM on viral replication and integration could be solidified. Problematically, IE1 is essential to promote HHV-6B replication in infected cells, and thus any IE1 knockdown (KD) or knockout (KO) approach will generate data that are hard to interpret. As mentioned by Reviewers 1 and 3, the ideal experiment to address this concern would be to infect cells with an HHV-6B virus in which IE1 contains a small truncation or a mutation that specifically suppresses its ability to inhibit ATM. Creating IE1 deletion and single mutants in the HHV-6 genome is technically challenging and can only be achieved using herpesvirus bacterial artificial chromosome (BAC)(Warden et al., 2011). Although HHV-6A BAC was previously described (Borenstein & Frenkel, 2009; Tang et al., 2010), our multiple attempts at generating HHV-6B BAC remained unsuccessful. As an alternative, we will investigate if the inhibition of ATM by using the ATM inhibitor (KU-55933) or its depletion by an shRNA, impact HHV-6B replication and integration as proposed by Reviewers 1 and 3. Specifically, MOLT-3 cells will be either treated with 10 µM KU-55933 or depleted for ATM with shATM(Rodier et al., 2009) prior to infection. DMSO and shLUC will be used as controls, respectively. These experiments will allow us to determine if ATM inhibition enhances HHV-6B replication and/or integration.

   1.2 Reviewer 2: "Although they have nicely mapped the interaction (between IE1 and NBS1), the authors have not yet defined the mechanism of ATM inhibition. They propose a number of possibilities in the discussion, but none are yet tested experimentally. The manuscript would be strengthened by further exploration of these possibilities. Does the sequence or proposed structure give any insights into interactions that could be relevant? Is IE1 phosphorylated by ATM, and could this affect the binding of other proteins?"

   We thank the Reviewer for pinpointing that a deeper characterization of the mechanism of ATM inhibition would allow us to support our model. In the manuscript, we discuss the possibility that IE1 inhibits ATM activation by preventing the interaction between the FxF/Y motif of NBS1 and ATM. Although we do not detect a strong interaction between IE1 and ATM (Fig. 5A), we have not yet investigated if the ATM-inhibitory domain (ATMiD) is required for IE1 to prevent the recruitment of ATM by NBS1 at the LacO array (Fig. 5E). Thus, we will determine if an ∆ATMiD IE1 inhibits the interaction between NBS1 and ATM in this assay. If the ATMiD domain interferes with the interaction of NBS1 with ATM, we expect to see no inhibition of NBS1 activation of ATM in cells that express 3xFlag-HHV-6B IE1 ∆ATMiD.

   Another possibility is that IE1 inhibits ATM activation indirectly by interacting with the nucleosome. The latter possibility is based on the finding that the C-terminal domain of HHV-5 IE1 contains an arginine-serine (RS) motif that interacts with the acidic patch of the nucleosome(Fang et al., 2016). Interestingly, HHV-6B IE1 sequence analysis reveals two RS motifs at positions 852-53 and 1033-34. Thus, the conserved RS residues (R852A/S853A and R1033A/S1034A) will be mutated in the ATMiD domain of HHV-6B IE1 (810-1078), and their ability to inhibit ATM activation will be quantified by immunofluorescence approach as described in Fig 6 D-E. In parallel, GST-tagged recombinant ATMiD of HHV-6B IE1 will be produced, and pulldown experiments will investigate their ability to bind to nucleosomes. We already have purified nucleosomes in the lab and have the expertise for this type of analysis(Galloy et al., 2021; Sitz et al., 2019).

   Thanks to the Reviewer's comment, we performed sequence analyses for putative ATM phosphorylation sites (SQ/TQ) and found that the protein contains 6 of them, two of which are in the ATMiD of the protein. To determine if the viral protein is a substrate of ATM, we will immunoprecipitate IE1 from MOLT-3 infected cells and use the well-characterized pSQ/pTQ antibody in western blotting analyses. The immunoprecipitation will be done in denaturing conditions to avoid interference with other endogenous interactors of IE1. If the protein is phosphorylated in an ATM-dependent manner, we will test the impact of these mutants on ATM inhibition as done in Fig. 6 D-E.

   Altogether, these experiments will allow us to refine our understanding of the mechanism by which HHV-6B IE1 inhibits ATM activation in host cells.

   1.3* Reviewer 2: "Could the effects of IE1 be linked to other post-translational modifications? The literature suggests this protein to be SUMOylated. Is SUMOylation relevant to the effects on ATM activation?" *

   The Reviewer is right. Our group showed that IE1 is sumoylated on K802R in a SUMO interacting motif (SIM)-dependent manner (V775, I776, V777)(Collin et al., 2020). In the LacO/LacR assays, we already showed that the K802R and SIM mutant (775AAA777) do not impact the interaction of IE1 with NBS1. Although the sumoylated site and the SIM lie outside of the ATMiD, we cannot rule out the possibility that this post-tranlationnal modification impacts ATM inhibition by IE1 throughout a conformational interference. To address this possibility, we will characterize the ability of the single and double K802R/SIM mutant proteins to inhibit the activation of ATM, as described in Fig 6 D-E.

   2 - ____Description of the revisions already incorporated in the transferred manuscript

   The following comments and all minor comments raised by the Reviewers have been incorporated into the transferred manuscript:

   2.1* Reviewer 2: "In Figure 1, they look at micronuclei formation but MNi is not defined the main text."*

   We thank the Reviewer for noticing this mistake. MNi is now defined as micronuclei in line 138.

   2.2* Reviewer 3: "As discussed by the authors, HHV-6B IE1 inhibits DSB signaling through NSB1, but we cannot know how this inhibition (might be increase genome instability of both host and virus) enhances viral replication and integration. The readers are easy to understand if the* authors described it in the discussion or analyzed by KD or KO of IE1 in infected cells."

   The Reviewer is right. We cannot rule out that increased genomic instability enhances viral replication. Thus, we add the following sentences to clarify this point in the discussion.

   Line 371-374: "Finally, the model presented here assumes that NBS1 and ATM activity must be inhibited to prevent their detrimental effect on viral replication. However, it is impossible to rule out that enhanced viral replication and integration result from the increased level of genomic instability induced in host cells upon viral infection. Further studies will be required to address this question."

   2.3* Reviewer 3: "Described in lines 354-356 are the case of lytic cycle only. In the lytic cycle, the infected cells will die soon after viral replication. and there is no chance to become tumor. However, the state of ciHHV-6 or latently infected cells can be affected by genome instability during IE1 expression. Please add discussion."*

   We thank the Reviewer for raising this important point. We agree that the real threat for the host cells regarding tumor development is genomic instability promoted by the expression of IE1 during latent infection or from an integrated form of the virus. Consistent with this possibility, our original manuscript contains this sentence in the abstract:

   Line 60-62: "Interestingly, as IE1 expression has been detected in cells of subjects with the inherited chromosomally-integrated form of HHV-6B (iciHHV-6B), a condition associated with several health conditions, our results raise the possibility of a link between genomic instability and the development of iciHHV-6-associated diseases."

   To further emphasize this point, the following sentence has now been added to the discussion:

   Line 349-356: During the lytic cycle, the accumulation of genomic instability in the host cell genome is not a problem as these cells will die upon the lysis provoked by the virus to release new virus particles. However, more selective inhibition of ATM by IE1 during the latent cycle of HHV-6B or from iciHHV-6B would avoid a detrimental accumulation of genomic alterations in host cells. This model would be consistent with the fact that HHV-6B is not associated with a higher frequency of cancer development, as would be expected if global DSB signaling was inhibited in these cells. Alternatively, expression of IE1 upon the exit of latency may inhibit global DSB signaling, but this phenomenon is restricted to the early stages of the process, thereby minimizing the impact on the host cell's genomic stability.

   2.4* Reviewer 3: Line 114, Miura et al (J Infect Dis 223:1717-1723 [2021]) should be cited.*

   This reference has been added in line 113. In the discussion, we also introduce the citation where we mention the link between HHV-6B integration and abortion, line 362 of the revised manuscript.

   3 - ____Description of the revisions that will not be carried out

   3.1* Reviewer 2: "Does it (HHV-6B IE1) also share other activities with herpesvirus proteins e.g. ubiquitinylation?"*

   IE1 shares very little sequence homology with proteins from other herpesviruses (except HHV-6A and HHV-7), meaning that deductions based on primary sequence analysis are very limited. Any attempt at understanding the function of HHV-6B IE1 by structure analysis prediction software did not predict any known function or domain. Thus, most of our knowledge of IE1 relies on experiments that used IE1 truncation (this study and (Jaworska et al., 2007)) and point mutants(Collin et al., 2020). The protein contains no conserved RING or HECT domain that would hint at an E3-ligase activity and does not share homology with other herpes proteins that promote ubiquitylation events, such as ICPO from HSV-1(Rodríguez et al., 2020). We believe that, at this point, there is not enough evidence to investigate further if HHV-6B IE1 has an E3-ligase activity.

   3.2* Reviewer 3: Lines 52, "Expression of immediate early protein 1 (IE1) was sufficient to recapitulate this phenotype" is not right. The authors showed that IE1 blocked ATM signaling in transient experiments but they did not show any evidence in infected cells. Kock down or Kock out of IE1 is important to conclude it."*

   We agree with the Reviewer HHV-6B IE1 knockdown, or knockout, would allow us to conclude that IE1 is the only protein to target DSB signaling in the infected cells. As mentioned by the Reviewer (see point 3.3 and 1.1), IE1 is essential to promote HHV-6B replication in infected cells. Thus, any knockdown or knockout approach will generate data that are hard to interpret. In contrast, the generation of an HHV-6 genome containing truncation or point mutation that abolishes its ability to inhibit ATM signaling should allow us to bypass this issue. While we believe this question is important, human resource shortages prevent us from addressing this point in an acceptable time frame. Instead, we propose investigating the role of ATM activity in HHV-6B replication and integration. We also rephrased the sentence highlighted by Reviewer 3:

   Line 51-52: "Expression of immediate early protein 1 (IE1) phenocopies this phenotype and blocks further homology-directed double-strand break (DSB) repair."

   3.3* Reviewer 3: The authors did not analyze the effect of viral manipulation as they did not analyze KO or KD of IE1. Even if HHV-6B IE1 is essential for viral replication, they can use dominant negative mutant of IE1 or NSB1 determined in this manuscript.*

   Reviewer is right. As discussed in points 3.2 and 1.1, we haven’t tried to rescue IE1 knockdown, or knockout in infected cells. Rescue experiments of IE1 by transient transfection of dominant negative IE1 mutant would require a high level of transfection in MOLT-3 cells and small truncation or mutations of IE1 that revert the ATM inhibitory function of IE1. Screening additional sets of truncations/mutants of IE1 that abolish its ability to inhibit ATM and optimizing the poor transfection efficiency of the lymphoid cell line MOLT-3 will take time and resources that we don’t have at this moment. Thus, we believe that this point should be addressed in follow-up studies.

   REFERENCES

   Borenstein, R., & Frenkel, N. (2009). Cloning human herpes virus 6A genome into bacterial artificial chromosomes and study of DNA replication intermediates. Proceedings of the National Academy of Sciences of the United States of America, 106(45). https://doi.org/10.1073/pnas.0908504106

   Collin, V., Gravel, A., Kaufer, B. B., & Flamand, L. (2020). The promyelocytic leukemia protein facilitates human herpesvirus 6B chromosomal integration, immediate-early 1 protein multiSUMOylation and its localization at telomeres. PLoS Pathogens, 16(7). https://doi.org/10.1371/journal.ppat.1008683

   Fang, Q., Chen, P., Wang, M., Fang, J., Yang, N., Li, G., & Xu, R.-M. (2016). Human cytomegalovirus IE1 protein alters the higher-order chromatin structure by targeting the acidic patch of the nucleosome. ELife, 5. https://doi.org/10.7554/elife.11911

   Galloy, M., Lachance, C., Cheng, X., Distéfano-Gagné, F., Côté, J., & Fradet-Turcotte. (2021). Approaches to study native chromatin-modifying activities and function. Frontiers in Cell and Developmental Biology, Section Epigenomics and Epigenetics, In Press.

   Jaworska, J., Gravel, A., Fink, K., Grandvaux, N., & Flamand, L. (2007). Inhibition of Transcription of the Beta Interferon Gene by the Human Herpesvirus 6 Immediate-Early 1 Protein. Journal of Virology, 81(11), 5737–5748. https://doi.org/10.1128/jvi.02443-06

   Rodier, F., Coppé, J. P., Patil, C. K., Hoeijmakers, W. A. M., Muñoz, D. P., Raza, S. R., Freund, A., Campeau, E., Davalos, A. R., & Campisi, J. (2009). Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion. Nature Cell Biology, 11(8). https://doi.org/10.1038/ncb1909

   Rodríguez, M. C., Dybas, J. M., Hughes, J., Weitzman, M. D., & Boutell, C. (2020). The HSV-1 ubiquitin ligase ICP0: Modifying the cellular proteome to promote infection. In Virus Research (Vol. 285). https://doi.org/10.1016/j.virusres.2020.198015

   Sitz, J., Blanchet, S. A. S. A., Gameiro, S. F. S. F., Biquand, E., Morgan, T. M. T. M., Galloy, M., Dessapt, J., Lavoie, E. G. E. G., Blondeau, A., Smith, B. C. B. C., Mymryk, J. S. J. S., Moody, C. A. C. A., & Fradet-Turcotte, A. (2019). Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response. Proceedings of the National Academy of Sciences of the United States of America, 116(39), 19552–19562. https://doi.org/10.1073/pnas.1906102116

   Tang, H., Kawabata, A., Yoshida, M., Oyaizu, H., Maeki, T., Yamanishi, K., & Mori, Y. (2010). Human herpesvirus 6 encoded glycoprotein Q1 gene is essential for virus growth. Virology, 407(2). https://doi.org/10.1016/j.virol.2010.08.018

   Warden, C., Tang, Q., & Zhu, H. (2011). Herpesvirus BACs: Past, present, and future. In Journal of Biomedicine and Biotechnology (Vol. 2011). https://doi.org/10.1155/2011/124595

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   Mar 7, 2023

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   Referee #3

   Evidence, reproducibility and clarity

   In this manuscript, the authors report that human herpesvirus 6B (HHV-6B) infection suppresses the host cell's ability to induce ATM-dependent signaling pathways. At least one of the viral proteins named IE1 block ATM signaling and further homology-directed double-strand break (DSB) repair in these cells. The ATM-dependent DNA damage response (DDR) is activated by infection of many viruses and suppresses their replications. Some of them induce the degradation of the MRE11/RAD50/NBS1 (MRN) complex and prevent subsequent DDR signaling. In the case of HHV-6B IE1, the N-terminal domain of it interacts with the MRN complex protein NBS1, …

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   Referee #3

   Evidence, reproducibility and clarity

   In this manuscript, the authors report that human herpesvirus 6B (HHV-6B) infection suppresses the host cell's ability to induce ATM-dependent signaling pathways. At least one of the viral proteins named IE1 block ATM signaling and further homology-directed double-strand break (DSB) repair in these cells. The ATM-dependent DNA damage response (DDR) is activated by infection of many viruses and suppresses their replications. Some of them induce the degradation of the MRE11/RAD50/NBS1 (MRN) complex and prevent subsequent DDR signaling. In the case of HHV-6B IE1, the N-terminal domain of it interacts with the MRN complex protein NBS1, the interaction of which might recruit IE1 to DSB and the C-terminal domain of IE1 inhibits ATM. The authors also showed that depletion of NBS1 enhanced HHV-6B replication. Viral integration of HHV-6B into the cellular chromosomes was enhanced by the NSB1 depletion in ATL-negative HeLa cells, supporting the models that the viral integration occurs via telomere elongation rather than through DNA repair.

   Major comments

   This manuscript is well written and will be of interest to the readers. The data seems convincing and statistical analysis is adequate. However, the role and significance of HHV-6B IE1 in infected cells was not analyzed well. If there are not analyzed, the data only show the role of the MRN complex or the only a single protein NSB1 for HHV-6B replication and they cannot conclude that HHV-6B IE1 hampers the ATM signaling for proper viral replication. I have a few comments listed below to improve this manuscript. All of them might be required for a couple of months.

   • (i) Lines 52, "Expression of immediate early protein 1 (IE1) was sufficient to recapitulate this phenotype" is not right. The authors showed that IE1 blocked ATM signaling in transient experiment but they did not show any evidence in infected cells. Kock down or Kock out of IE1 is important to conclude it.
   • (ii) In Fig7, the role of the other factors in the ATM-dependent DDR (such as ATM) should be analyzed by knock down or inhibitors.
   • (iii) The authors did not analyze the effect of viral manipulation as thy did not analyze KO or KD of IE1. Even if HHV-6B IE1 is essential for viral replication, they can use dominant negative mutant of IE1 or NSB1 determined in this manuscript.
   • (iv) As discussed by the authors, HHV-6B IE1 inhibit DSB signaling through NSB1, but we cannot know how this inhibition (might be increase genome instability of both host and virus) enhances viral replication and integration. The readers are easy to understand if the authors described it in the discussion or analyzed by KD or KO of IE1 in infected cells.

   Minor comments

   • (i) Described in lines 354-356 are the case of lytic cycle only. In the lytic cycle, the infected cells will die soon after viral replication. and there is no chance to become tumor. However, the state of ciHHV-6 or latently infected cells can be affected by genome instability during IE1 expression. Please add discussion.
   • (ii) Line114, Miura et al (J Infect Dis 223:1717-1723 [2021]) should be cited.

   Significance

   HHV-6B is ubiquitous herpesvirus which cause exanthem subitem and encephalitis, although effective antiviral is not established yet. Characteristically, HHV-6B has ability to integrate its genome into host. How HHV-6B replicate and integrate its genome in host cells is one of the most important question in this field. I am basic virologist mainly focusing on this virus and believe this manuscript includes important notion for our field.

   To counteract ATM-mediated signaling, many viruses induce the degradation of the MRN complex and prevent subsequent DDR signaling. The mechanism of HHV-6B IE1 described in this manuscript is unique and might be interested by the readers from many fields.

   Furthermore, around 1% of human populations harbor chromosomally integrated HHV-6B in their genome. The pathogenesis of it is not completely understand but must be important not only for virologist but also all of us.

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   Referee #2

   Evidence, reproducibility and clarity

   Viruses have evolved different strategies by which they manipulate the host DNA damage response (DDR) in order to propagate in infected cells. This study shows how the human herpesvirus 6B (HHV-6B) blocks homology-directed double-stranded DNA break repair by the immediate early protein 1 (IE1) which they demonstrate inhibits the host ATM kinase. They employ microscopy and cytometry approaches to probe genomic instability, signaling, and interactions between virus and host. They use infection of MOLT-3 cells and induction of IE1 in U2OS cells to examine these mechanisms and the effects on genome stability. They show inhibition of …

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   Referee #2

   Evidence, reproducibility and clarity

   Viruses have evolved different strategies by which they manipulate the host DNA damage response (DDR) in order to propagate in infected cells. This study shows how the human herpesvirus 6B (HHV-6B) blocks homology-directed double-stranded DNA break repair by the immediate early protein 1 (IE1) which they demonstrate inhibits the host ATM kinase. They employ microscopy and cytometry approaches to probe genomic instability, signaling, and interactions between virus and host. They use infection of MOLT-3 cells and induction of IE1 in U2OS cells to examine these mechanisms and the effects on genome stability. They show inhibition of H2AX phosphorylation, and inhibition of homology-directed repair with reporter assays. They discovered that IE1 interacts with the cellular NBS1 protein, localizes to DNA breaks, and inhibits activation of ATM kinase. They map two distinct domains that promote NBS1 interaction and the inhibition of ATM activation. They show that depletion of NBS1 promotes lytic replication in MOLT-3 cells, and also decreases the frequency of integration, at least in some semi-permissive cells.

   Major:

   1. Although they have nicely mapped the interactions, the authors have not yet defined the mechanism of ATM inhibition. They propose a number of possibilities in the Discussion but none are yet tested experimentally. The manuscript would be strengthened by further exploration of these possibilities. Does the sequence or proposed structure give any insights into interactions that could be relevant? Is IE1 phosphorylated by ATM and could this affect binding of other proteins?
   2. Could the effects of IE1 be linked to other post-translational modifications? The literature suggests this protein to be SUMOylated. Is SUMOylation relevant to the effects on ATM activation? Does it also share other activities with herpesvirus proteins e.g. ubiquitinylation?
   3. Are the effects on the lifecycle (lytic replication and integration) affected by ATM kinase in the same way as NBS1?

   Minor:

   1. In Figure 1 they look at micronuclei formation but MNi is not defined the main text.

   Significance

   Overall, the manuscript is well written the experiments are performed in a rigorous manner, and the biology uncovered is of broad scientific interest. It is now known that a number of DNA viruses inhibit aspects of the cellular DNA sensing and repair machinery to overcome antiviral responses and promote infection. Understanding how this achieved by different viral systems provides insights into cellular DNA damage signaling and repair. It also informs about how viruses can trigger genomic instability. In this case, the authors have uncovered a novel way that the ATM kinase is inhibited during HHV-6B infection by the IE1 protein. They show that HHV-6B infection induces genomic instability. Integration of the HHV-6 genome results in inherited chromosomally-integrated (ici)HHV-6A/B. They have some data to show that virus replication is inhibited by NBS1 and that viral integration may be partially impacted. These results have implications for understanding viral integration and genomic instability with this human pathogen. They advance the field and expand our understanding of how viruses manipulate repair pathways and lead to genomic instability. Strengths include the rigorous analysis of interactions with IE1 and impacts on cellular pathways. Limitations include the lack of mechanism for inhibition and the weaker links to viral biology. The results will be on interest to those studying virus-host interactions as well as those studying repair pathways beyond virus infection.

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   Referee #1

   Evidence, reproducibility and clarity

   In this manuscript Collin and colleagues found that the human herpesvirus 6B (HHV-6B) causes genomic instability in host cells by suppressing the host cell's ability to induce ATM-dependent signaling pathways. The authors show that the immediate early protein 1 (IE1) of HHV-6B is sufficient to block homology-directed double-strand break (DSB) repair and ATM-mediated DNA damage signaling. Interestingly, the authors show that IE1 does not affect the stability of the MRN complex, but instead uses two distinct domains to inhibit ATM activation. Finally, the authors show that suppression of NBS1 is critical for the ability of HHV-6B to …

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   Referee #1

   Evidence, reproducibility and clarity

   In this manuscript Collin and colleagues found that the human herpesvirus 6B (HHV-6B) causes genomic instability in host cells by suppressing the host cell's ability to induce ATM-dependent signaling pathways. The authors show that the immediate early protein 1 (IE1) of HHV-6B is sufficient to block homology-directed double-strand break (DSB) repair and ATM-mediated DNA damage signaling. Interestingly, the authors show that IE1 does not affect the stability of the MRN complex, but instead uses two distinct domains to inhibit ATM activation. Finally, the authors show that suppression of NBS1 is critical for the ability of HHV-6B to replicate in permissive cells. In contrast, suppression of NBS1 increases the rate of integration in semi permissive cells. Overall, this study provides a mechanistic insight into HHV-6B infection and viral integration into telomeres may promote genomic instability and the development of certain diseases associated with inherited chromosomally integrated form of HHV-6B.

   Significance

   Overall, this is a superb manuscript, the data are clear, well controlled, and well presented. This reviewer has only a minor suggestion/ comment.

   The authors show convincingly that E1a can bind NBS1 and suppress ATM activation. However, it is not clear whether suppression of ATM is critical for HHV-6 replication. The ideal experiment would be an infection with a virus depleted of E1A (or expressing a defective E1A mutant). I realize that this would be a challenging experiment. An alternative experiment would be to test whether suppression of ATM has the same effect on HHV-6 replication and integration as NBS1 depletion.

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   Version published to 10.1101/2021.07.31.454588v2 on bioRxiv

   Mar 7, 2023
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   Version published to 10.1101/2021.07.31.454588v1 on bioRxiv

   Jul 31, 2021