Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies
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Curated by eLife
Evaluation Summary:
This work has potential broad interest to both virologists and cell biologists interested in the regulatory functions of promyelocytic leukaemia nuclear bodies (PML-NBs). The authors use a combination of imaging techniques to identify PML, the principal scaffolding protein of PML-NBs, to form a variety of different structures in response to viral infection, immune stimulation, and DNA damage. Thestudy identifies PML to restrict the replication of human cytomegalovirus (HCMV) at multiple stages of infection through the formation of alternate PML-scaffold assemblies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
PML nuclear bodies (PML-NBs) are dynamic interchromosomal macromolecular complexes implicated in epigenetic regulation as well as antiviral defense. During herpesvirus infection, PML-NBs induce epigenetic silencing of viral genomes, however, this defense is antagonized by viral regulatory proteins such as IE1 of human cytomegalovirus (HCMV). Here, we show that PML-NBs undergo a drastic rearrangement into highly enlarged PML cages upon infection with IE1-deficient HCMV. Importantly, our results demonstrate that dual signaling by interferon and DNA damage response is required to elicit giant PML-NBs. DNA labeling revealed that invading HCMV genomes are entrapped inside PML-NBs and remain stably associated with PML cages in a transcriptionally repressed state. Intriguingly, by correlative light and transmission electron microscopy (EM), we observed that PML cages also entrap newly assembled viral capsids demonstrating a second defense layer in cells with incomplete first-line response. Further characterization by 3D EM showed that hundreds of viral capsids are tightly packed into several layers of fibrous PML. Overall, our data indicate that giant PML-NBs arise via combined interferon and DNA damage signaling which triggers entrapment of both nucleic acids and proteinaceous components. This represents a multilayered defense strategy to act in a cytoprotective manner and to combat viral infections.
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Author Response
Reviewer #1 (Public Review):
The entrapment of viral DNA and viral capsids in PML cages is efficiently achieved only when the cells are infected with a low MOI (ie one copy of HCMV). This is a well performed work, which describes an interesting pattern of nuclear changes upon viral infection. However, the question that remains is how is PML capable of sensing the number of viral genomes. As PML cages can be formed in the absence of viral infection, exemplified by the fact that IFN signalling and DNA damage can induce their formation, I would expect the authors to deepen this part of the work, which is rather limited to data presented in Figure 4. The functional relevance of disruption of PML cages by viral protein IE1 and its impact on HCMV replication (shown in Figure 6) is a nice demonstration of the strategy that
Author Response
Reviewer #1 (Public Review):
The entrapment of viral DNA and viral capsids in PML cages is efficiently achieved only when the cells are infected with a low MOI (ie one copy of HCMV). This is a well performed work, which describes an interesting pattern of nuclear changes upon viral infection. However, the question that remains is how is PML capable of sensing the number of viral genomes. As PML cages can be formed in the absence of viral infection, exemplified by the fact that IFN signalling and DNA damage can induce their formation, I would expect the authors to deepen this part of the work, which is rather limited to data presented in Figure 4. The functional relevance of disruption of PML cages by viral protein IE1 and its impact on HCMV replication (shown in Figure 6) is a nice demonstration of the strategy that virus evolved to disrupt these structures. It would also be very interesting to understand how are these structures metabolized in the absence of viral infection, especially because the authors highlight the importance of IFN and DNA damage for their formation.
We thank reviewer #1 for the positive comments on our work. We show that induction of DNA damage signaling together with interferon treatment is sufficient to induce PML cages and we now provide new evidence to demonstrate that PML cages co-localize with markers of DNA damage both after doxorubicin treatment and after infection with HCMV∆hIE1. Furthermore, we observed that treatment of HCMV∆hIE1 infected cells with an ATM inhibitor interferes with PML cage formation providing further evidence for a requirement of DNA damage signaling for formation of these structures. We assume that PML itself does not serve as the main sensor of viral genomes. This can be deduced from the fact that only a minority of PML-NBs associate with viral genomes in infected cells. We agree with reviewer #1 that the question of how cells sense the number of viral genomes is highly interesting, however, we feel that this is beyond the scope of this manuscript. Most probably, HCMV genomes are sensed by a cellular factor different from PML that is present in low amounts thus explaining the rapid saturation of PML mediated defense. It would of course also be interesting to exactly understand the metabolization of giant PML-NBs. So far, we know that treatment of cells with arsenic trioxide not only degrades normal PML-NBs but also giant PML-NBs. This suggests that RNF4 is also responsible for metabolization of giant PML-NBs.
Reviewer #2 (Public Review):
Utilising a combination of high-resolution light microscopy and electron microscopy imaging, Scherer et al., identify promyelocytic leukaemia nuclear bodies (PML-NBs) to undergo extensive rearrangement during HCMV infection in the absence of the viral PML- antagonist IE1 protein. These data identify PML, the principal scaffolding protein of PML-NBs, to undergo dynamic structural rearrangements throughout the course of HCMV infection in a manner dependent on the activation of IFN-mediated innate immune defences and induction of the cellular DNA damage response (DDR). As such, the authors identify PML to play sequential roles in the spatiotemporal restriction of HCMV at multiple phases of infection dependent on the immunological state of the cell. The manuscript is accessible and well laid out, exceptionally well written, and experiments conducted to a high standard. The authors conclusions are generally supported by the data without over interpretation. Some aspects of the image analysis, including population and statistical testing, and DDR activation during infection require extending to add support to their major conclusions. Nevertheless, the study overall resolves many conflicting issues in the current literature surrounding the antiviral properties of PML during HCMV infection and identifies important future areas of research pertinent to both virology, immunology, and cell biology research communities. Fascinating science!
We thank reviewer #2 for the positive comments. As suggested by reviewer #2, we performed several new experiments addressing DDR activation during infection. We also repeated several of the experiments to be able to include population analysis and statistical testing.
Reviewer #3 (Public Review):
• Summary. In their study Scherer et al. demonstrate that the PML NBs act as a nuclear intrinsic antiviral response against the incoming HCMV genomes and more surprisingly against capsids. Using a set of approaches such as click chemistry to label the viral genomes, immunofluorescence, and electron microscopy their show that PML NBs entrap incoming viral genomes forming giant PML NBs leading to transcriptionally repressed viral genomes. If viral genomes escape the first layer of restriction activity of the PML NBs, to progress into the lytic cycle, they show that the PML NBs are also able to entrap capsids in a second layer of antiviral defense mechanism. Finally, they show that PML cages formation containing viral genomes or nucleocapsids arise via the combined interferon and DNA damage signaling.
• Major strengths and weaknesses.
Strengths
- The study nicely demonstrates the major involvement of the PML protein and PML NBs in the control of the incoming viral genomes during infection by the human cytomegalovirus (HCMV).
- Results are clear, nicely illustrated and presented in a easily understandable manner.
- The methods in use especially click chemistry to visualize the incoming viral genomes and combination of light microscopy with CLEM and FIB-SEM to visualize viral capsids entrapment in the nucleus are really challenging.
- The study is of broad interest regarding various pathological situations whether they result from viral infection (HCMV, HSV, VZV, HPV, HBV...) or genetic disorders (ICF, ALT), and in which PML NBs play major roles.
Weakness
- Although at mechanistical and molecular levels the study does not suffer of major weaknesses to reviewer's opinion, at the physiological level it would have been interesting to provide some data on the formation of PML cages in cells supporting HCMV latent infection such as bone marrow CD34+ cells or alternatively THP1 cells. However, the reviewer acknowledges the fact that this could be out of the scoop of this study given the amount of work it could necessitate to provide a complete set of data in cells supporting HCMV latency in physiological conditions.
Appraisal.
To reviewer's view there is no doubt that the authors achieved their aims to demonstrate the physical interaction between viral genomes and capsids with PML NBs and the role of this epigenetic regulation in the establishment and maintenance of HCMV latency. As such, they data nicely support previous studies either showing entrapment of incoming viral genomes during HSV-1 lytic infections and latency (Everett et al, 2007; Catez et al, 2012; Alandijany et al, 2018; Cohen et al, 2018), and of capsids during VZV infection (Reichelt et al, 2011, 2012). One of the originality of the study by Scherer et al. stands in the fact that it is the first time that such interactions are described for a herpesvirus of a subfamily other than alphaherpesvirinae (HCMV being a betaherpesvirus). Additionally, it is the first time that both, PML cages entrapping viral genomes or capsids are described for the same virus and during the process of infection.
References
Alandijany T, Roberts APE, Conn KL, Loney C, McFarlane S, Orr A & Boutell C (2018) Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection. PLoS Pathog 14: e1006769 Catez F, Picard C, Held K, Gross S, Rousseau A, Theil D, Sawtell N, Labetoulle M & LOMONTE P (2012) HSV-1 Genome Subnuclear Positioning and Associations with Host-Cell PML-NBs and Centromeres Regulate LAT Locus Transcription during Latency in Neurons. PLoS Pathog 8: e1002852 Cohen C, Corpet A, Roubille S, Maroui M-A, Poccardi N, Rousseau A, Kleijwegt C, Binda O, Texier P, Sawtell N, et al (2018) Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis. PLoS Pathog 14: e1007313 Everett RD, Murray J, Orr A & Preston CM (2007) Herpes simplex virus type 1 genomes are associated with ND10 nuclear substructures in quiescently infected human fibroblasts. 81 Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I & Arvin AM (2012) 3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography. PLoS Pathog 8: e1002740 Reichelt M, Wang L, Sommer M, Perrino J, Nour AM, Sen N, Baiker A, Zerboni L & Arvin AM (2011) Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus. PLoS Pathog 7: e1001266
Likely impact.
In the field of herpesviruses and other DNA and nuclear replicating viruses the role of PML NBs as part of the intrinsic immunity has become a major subject of investigations for several years. Hence, this work represents a major contribution in the understanding of the role of PML NBs in the antiviral response.
Additional context.
As investigated by the authors in figure 6 this work could be of interest for scientists working in the field of telomeres biology particularly in the context of cancer cells that maintain telomeres length by the alternative telomere lengthening (ALT) process. Indeed, in those cells telomeres are entrapped in PML NBs forming structures called ALT associated PML NBs (APBs). Any kind of study investigating similar behavior for PML NBs in sequestrating chromatin loci, whatever it is for HCMV, HSV, or other type of viruses are likely to bring new clues and idea concerning the role and the formation of the APBs.
We thank reviewer #3 for the positive comments on our manuscript. Reviewer #3 suggested that the paper should provide some data on the formation of PML cages in cells supporting HCMV latent infection such as bone marrow CD34+ cells or alternatively THP1 cells. Using THP1 cells as a latency model, we have previously demonstrated that PML does not serve as a key determinant for the establishment of HCMV latency (Wagenknecht et al., Viruses 2015). Rather, PMLNB proteins may act as cellular restriction factors during the dynamic process of viral reactivation. Since HCMV reactivation from latency is a rare event, it will be very challenging to study PML cage formation in the respective cells undergoing reactivation. We agree with reviewer #3 that this is beyond the scope of this manuscript.
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Evaluation Summary:
This work has potential broad interest to both virologists and cell biologists interested in the regulatory functions of promyelocytic leukaemia nuclear bodies (PML-NBs). The authors use a combination of imaging techniques to identify PML, the principal scaffolding protein of PML-NBs, to form a variety of different structures in response to viral infection, immune stimulation, and DNA damage. Thestudy identifies PML to restrict the replication of human cytomegalovirus (HCMV) at multiple stages of infection through the formation of alternate PML-scaffold assemblies.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The entrapment of viral DNA and viral capsids in PML cages is efficiently achieved only when the cells are infected with a low MOI (ie one copy of HCMV). This is a well performed work, which describes an interesting pattern of nuclear changes upon viral infection. However, the question that remains is how is PML capable of sensing the number of viral genomes. As PML cages can be formed in the absence of viral infection, exemplified by the fact that IFN signalling and DNA damage can induce their formation, I would expect the authors to deepen this part of the work, which is rather limited to data presented in Figure 4. The functional relevance of disruption of PML cages by viral protein IE1 and its impact on HCMV replication (shown in Figure 6) is a nice demonstration of the strategy that virus evolved to …
Reviewer #1 (Public Review):
The entrapment of viral DNA and viral capsids in PML cages is efficiently achieved only when the cells are infected with a low MOI (ie one copy of HCMV). This is a well performed work, which describes an interesting pattern of nuclear changes upon viral infection. However, the question that remains is how is PML capable of sensing the number of viral genomes. As PML cages can be formed in the absence of viral infection, exemplified by the fact that IFN signalling and DNA damage can induce their formation, I would expect the authors to deepen this part of the work, which is rather limited to data presented in Figure 4. The functional relevance of disruption of PML cages by viral protein IE1 and its impact on HCMV replication (shown in Figure 6) is a nice demonstration of the strategy that virus evolved to disrupt these structures. It would also be very interesting to understand how are these structures metabolized in the absence of viral infection, especially because the authors highlight the importance of IFN and DNA damage for their formation.
-
Reviewer #2 (Public Review):
Utilising a combination of high-resolution light microscopy and electron microscopy imaging, Scherer et al., identify promyelocytic leukaemia nuclear bodies (PML-NBs) to undergo extensive rearrangement during HCMV infection in the absence of the viral PML- antagonist IE1 protein. These data identify PML, the principal scaffolding protein of PML-NBs, to undergo dynamic structural rearrangements throughout the course of HCMV infection in a manner dependent on the activation of IFN-mediated innate immune defences and induction of the cellular DNA damage response (DDR). As such, the authors identify PML to play sequential roles in the spatiotemporal restriction of HCMV at multiple phases of infection dependent on the immunological state of the cell. The manuscript is accessible and well laid out, exceptionally …
Reviewer #2 (Public Review):
Utilising a combination of high-resolution light microscopy and electron microscopy imaging, Scherer et al., identify promyelocytic leukaemia nuclear bodies (PML-NBs) to undergo extensive rearrangement during HCMV infection in the absence of the viral PML- antagonist IE1 protein. These data identify PML, the principal scaffolding protein of PML-NBs, to undergo dynamic structural rearrangements throughout the course of HCMV infection in a manner dependent on the activation of IFN-mediated innate immune defences and induction of the cellular DNA damage response (DDR). As such, the authors identify PML to play sequential roles in the spatiotemporal restriction of HCMV at multiple phases of infection dependent on the immunological state of the cell. The manuscript is accessible and well laid out, exceptionally well written, and experiments conducted to a high standard. The authors conclusions are generally supported by the data without over interpretation. Some aspects of the image analysis, including population and statistical testing, and DDR activation during infection require extending to add support to their major conclusions. Nevertheless, the study overall resolves many conflicting issues in the current literature surrounding the antiviral properties of PML during HCMV infection and identifies important future areas of research pertinent to both virology, immunology, and cell biology research communities. Fascinating science!
-
Reviewer #3 (Public Review):
In their study Scherer et al. demonstrate that the PML NBs act as a nuclear intrinsic antiviral response against the incoming HCMV genomes and more surprisingly against capsids. Using a set of approaches such as click chemistry to label the viral genomes, immunofluorescence, and electron microscopy their show that PML NBs entrap incoming viral genomes forming giant PML NBs leading to transcriptionally repressed viral genomes. If viral genomes escape the first layer of restriction activity of the PML NBs, to progress into the lytic cycle, they show that the PML NBs are also able to entrap capsids in a second layer of antiviral defense mechanism. Finally, they show that PML cages formation containing viral genomes or nucleocapsids arise via the combined interferon and DNA damage signaling.
Major strengths and …
Reviewer #3 (Public Review):
In their study Scherer et al. demonstrate that the PML NBs act as a nuclear intrinsic antiviral response against the incoming HCMV genomes and more surprisingly against capsids. Using a set of approaches such as click chemistry to label the viral genomes, immunofluorescence, and electron microscopy their show that PML NBs entrap incoming viral genomes forming giant PML NBs leading to transcriptionally repressed viral genomes. If viral genomes escape the first layer of restriction activity of the PML NBs, to progress into the lytic cycle, they show that the PML NBs are also able to entrap capsids in a second layer of antiviral defense mechanism. Finally, they show that PML cages formation containing viral genomes or nucleocapsids arise via the combined interferon and DNA damage signaling.
Major strengths and weaknesses:
Strengths
- The study nicely demonstrates the major involvement of the PML protein and PML NBs in the control of the incoming viral genomes during infection by the human cytomegalovirus (HCMV).
- Results are clear, nicely illustrated and presented in a easily understandable manner.
- The methods in use especially click chemistry to visualize the incoming viral genomes and combination of light microscopy with CLEM and FIB-SEM to visualize viral capsids entrapment in the nucleus are really challenging.
- The study is of broad interest regarding various pathological situations whether they result from viral infection (HCMV, HSV, VZV, HPV, HBV...) or genetic disorders (ICF, ALT), and in which PML NBs play major roles.Weakness
- Although at mechanistical and molecular levels the study does not suffer of major weaknesses to reviewer's opinion, at the physiological level it would have been interesting to provide some data on the formation of PML cages in cells supporting HCMV latent infection such as bone marrow CD34+ cells or alternatively THP1 cells. However, the reviewer acknowledges the fact that this could be out of the scoop of this study given the amount of work it could necessitate to provide a complete set of data in cells supporting HCMV latency in physiological conditions.Appraisal:
To reviewer's view there is no doubt that the authors achieved their aims to demonstrate the physical interaction between viral genomes and capsids with PML NBs and the role of this epigenetic regulation in the establishment and maintenance of HCMV latency. As such, they data nicely support previous studies either showing entrapment of incoming viral genomes during HSV-1 lytic infections and latency (Everett et al, 2007; Catez et al, 2012; Alandijany et al, 2018; Cohen et al, 2018), and of capsids during VZV infection (Reichelt et al, 2011, 2012). One of the originality of the study by Scherer et al. stands in the fact that it is the first time that such interactions are described for a herpesvirus of a subfamily other than alphaherpesvirinae (HCMV being a betaherpesvirus). Additionally, it is the first time that both, PML cages entrapping viral genomes or capsids are described for the same virus and during the process of infection.
References
Alandijany T, Roberts APE, Conn KL, Loney C, McFarlane S, Orr A & Boutell C (2018) Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection. PLoS Pathog 14: e1006769
Catez F, Picard C, Held K, Gross S, Rousseau A, Theil D, Sawtell N, Labetoulle M & LOMONTE P (2012) HSV-1 Genome Subnuclear Positioning and Associations with Host-Cell PML-NBs and Centromeres Regulate LAT Locus Transcription during Latency in Neurons. PLoS Pathog 8: e1002852
Cohen C, Corpet A, Roubille S, Maroui M-A, Poccardi N, Rousseau A, Kleijwegt C, Binda O, Texier P, Sawtell N, et al (2018) Promyelocytic leukemia (PML) nuclear bodies (NBs) induce latent/quiescent HSV-1 genomes chromatinization through a PML NB/Histone H3.3/H3.3 Chaperone Axis. PLoS Pathog 14: e1007313
Everett RD, Murray J, Orr A & Preston CM (2007) Herpes simplex virus type 1 genomes are associated with ND10 nuclear substructures in quiescently infected human fibroblasts. 81
Reichelt M, Joubert L, Perrino J, Koh AL, Phanwar I & Arvin AM (2012) 3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography. PLoS Pathog 8: e1002740
Reichelt M, Wang L, Sommer M, Perrino J, Nour AM, Sen N, Baiker A, Zerboni L & Arvin AM (2011) Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus. PLoS Pathog 7: e1001266Likely impact:
In the field of herpesviruses and other DNA and nuclear replicating viruses the role of PML NBs as part of the intrinsic immunity has become a major subject of investigations for several years. Hence, this work represents a major contribution in the understanding of the role of PML NBs in the antiviral response.
Additional context:
As investigated by the authors in figure 6 this work could be of interest for scientists working in the field of telomeres biology particularly in the context of cancer cells that maintain telomeres length by the alternative telomere lengthening (ALT) process. Indeed, in those cells telomeres are entrapped in PML NBs forming structures called ALT associated PML NBs (APBs). Any kind of study investigating similar behavior for PML NBs in sequestrating chromatin loci, whatever it is for HCMV, HSV, or other type of viruses are likely to bring new clues and idea concerning the role and the formation of the APBs.
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