In order to establish a successful infection, viruses have to overcome intrinsic, innate, and adaptive host defenses which act in a cooperative manner to block viral replication and spread. The first line of intracellular defense is provided by intrinsic immune effectors that, in contrast to classical innate responses like the interferon system, are constitutively expressed and do not require pathogen-induced activation. Studies over the last 25 years have identified promyelocytic leukemia protein nuclear bodies (PML-NBs), also known as nuclear domain 10, as key mediators of intrinsic immunity against viruses from different families (Tavalai and Stamminger, 2008; Everett and Chelbi-Alix, 2007). These highly dynamic protein complexes are located in the interchromosomal space of the cell nucleus and appear as discrete foci with a size of 0.2–1.0 µm in diameter and a number of 1–30 PML-NBs per nucleus, depending on cell type and condition (Bernardi and Pandolfi, 2007). PML, the structure-defining component of PML-NBs, belongs to the tripartite motif (TRIM) protein family and is expressed in at least seven isoforms whose common N-terminal region consists of a RING domain, one or two B-boxes, and a coiled-coil (CC) domain. All PML isoforms are subject to covalent modification with small ubiquitin-like modifier (SUMO) proteins, which enables the recruitment of further components and is therefore essential for PML-NB biogenesis. Due to the high number of permanently or transiently recruited proteins, PML-NBs have been implicated in a variety of cellular processes including transcriptional regulation, control of apoptosis and cellular senescence as well as DNA damage response. Furthermore, the early observation that PML-NBs target invading genomes of several DNA viruses, including the herpesviruses herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV), papillomaviruses and adenoviruses, raised the concept that PML-NBs act as sites for deposition of viral DNA (Ishov and Maul, 1996; Ishov et al., 1997; Day et al., 2004). More recent research on HSV-1 has confirmed the nuclear association with parental viral genomes and has shown that HSV-1 DNA is enveloped by PML-NBs, which thereby contribute to the control of latency in infected neurons and to intrinsic restriction of lytic HSV-1 infection (Catez et al., 2012; Everett et al., 2007; Alandijany et al., 2018). In contrast, a study on the interplay of PML-NBs with adenoviral genomes has found a viral DNA replication factor but not genome complexes colocalizing with PML-NBs, thus arguing against a general role as deposition site for viral genomes (Komatsu et al., 2016).

Characterization of the intrinsic immune function of PML-NBs during herpesvirus infection has identified the major components PML, Sp100, Daxx, and ATRX as independent restriction factors that induce epigenetic silencing of viral DNA by recruiting chromatin-modifying enzymes (Tavalai and Stamminger, 2009). This restrictive activity enables PML-NBs to block one of the first steps in the herpesviral life cycle. However, it is saturable and can be overcome by high doses of virus. A different antiviral mechanism, acting on a later stage of infection, has been shown to affect the herpesvirus varicella-zoster virus (VZV). During VZV infection, PML-NBs target and enclose viral nucleocapsids, mediated through a specific interaction of PML isoform IV with the ORF23 capsid protein (Reichelt et al., 2011; Reichelt et al., 2012). In addition to their role in intrinsic immunity, accumulating evidence implicates PML-NBs in the innate immune defense. An interplay between PML-NBs and innate immunity has been discovered with the observation that interferon (IFN) treatment induces the expression of specific PML-NB factors, such as PML and Sp100, and leads to an increased size and number of foci (Chelbi-Alix et al., 1995; Lavau et al., 1995). In line with this, PML-NBs participate in the establishment of an IFN-induced antiviral state and depletion of PML reduces the capacity of IFNs to protect from viral infections (Regad et al., 2001; Chee et al., 2003). Recent evidence has found that PML itself acts as a co-regulatory factor for the induction of IFN-stimulated genes, suggesting an even closer cross talk between intrinsic and innate immune mechanisms (Ulbricht et al., 2012; Kim and Ahn, 2015; El Asmi et al., 2014; Scherer et al., 2016).

In light of the broad antiviral activity of PML-NBs, it is not surprising that viruses encode antagonistic effector proteins that employ diverse strategies to inactivate single PML-NB proteins or disrupt the integrity of the whole structure. HCMV, a ubiquitous beta-herpesvirus causing serious disease in immunocompromised individuals, encodes at least two effector proteins that act in a sequential manner to efficiently antagonize PML-NB-based repression. Upon infection, the tegument-delivered protein pp71 is imported into the nucleus where it leads to dissociation of ATRX from PML-NBs, followed by proteasomal degradation of Daxx (Lukashchuk et al., 2008; Saffert and Kalejta, 2006). As this facilitates initiation of viral immediate-early gene expression, the immediate-early protein 1 (IE1) can be expressed and induces a complete dispersal of PML-NBs within the first hours of infection. Mechanistic studies have revealed that IE1 directly interacts with PML through its all alpha-helical core domain and blocks de novo SUMOylation of PML, thus disrupting PML-NB integrity (Schilling et al., 2017). While the PML-antagonistic protein ICP0 of HSV-1 has been shown to induce a widespread degradation of SUMO-modified proteins, IE1 uses a more specific, but yet not fully elucidated mechanism to inhibit PML SUMOylation and thereby promote viral replication (Boutell et al., 2011). Due to such rapid and effective countermeasures, recombinant viruses that lack antagonistic proteins provide a valuable tool to study antiviral activities of cellular restriction factors, which would otherwise not be detectable.

Here, we report an interferon- and DNA damage signaling-induced formation of huge PML spheres, referred to as PML cages, which occurs during infection with IE1-deleted HCMV. Visualization of viral DNA with clickable fluorescent azides revealed that input HCMV genomes are entrapped by PML-NBs and remain stably encased by PML cages, leading to repression of viral gene expression as a first layer of antiviral defense. Moreover, we identify a second layer of PML-based protection for cells escaping the gene silencing-driven defense: we demonstrate that PML cages entrap newly assembled HCMV capsids in late infected cells by using correlative light and transmission electron microcopy (CLEM). 3D reconstruction of PML cages after focused ion beam-scanning electron microscopy (FIB-SEM) tomography illustrates hundreds of HCMV capsids sequestered by fibrous PML structures. Overall, these data indicate a dual, PML-based inhibition of HCMV infection and suggest entrapment of viral material as a general restriction mechanism used by PML-NBs.

Since their discovery, PML-NBs have been topic of intense investigation, both to elucidate the structure and to understand the function of these enigmatic nuclear organelles. The general interest in PML-NBs originated from the tight link between PML-NB integrity and several pathological conditions such as acute promyelocytic leukemia (APL), neurodegenerative diseases as well as viral infections, and resulted in a large number of publications implicating PML-NBs in diverse cellular processes (Tavalai and Stamminger, 2008; Grimwade and Solomon, 1997; Yasuda et al., 1999). These different biochemical activities likely arise from the high number of PML-associated proteins as well as the dynamic nature of PML-NBs, which differ in composition, number, size, and position depending on the cellular state (Eskiw et al., 2003; Weidtkamp-Peters et al., 2008). Here, we describe unusually large PML-NBs (Figure 1), referred to as PML cages, which have the capacity to entrap both parental viral genomes and newly assembled viral capsids thus contributing to the restriction of human cytomegalovirus infection. Concerning biogenesis and composition, PML cages are similar to normal PML-NBs, as the PML protein functions as a key regulator (Figure 2b) that recruits all other main components with the only exception of ATRX (Figure 2a). Under physiological conditions, ATRX is recruited to PML-NBs via an interaction with the Daxx protein in order to form a chromatin remodeling complex (Tang et al., 2004). Upon HCMV infection, Daxx is initially targeted for degradation by the viral tegument protein pp71; however, it re-accumulates at later stages of infection and can be detected at PML cages (Figure 2a, Saffert and Kalejta, 2006). Since several interaction partners of Daxx, including ATRX, have been shown to bind in a mutually exclusive manner, the absence of ATRX from PML cages may indicate an altered regulation of Daxx during HCMV infection that leads to a switch of interaction partners (Li et al., 2017).

In addition to the main PML-NB components, we found heterochromatin protein HP1α localizing to PML cages and being recruited by Sp100 (Figure 6a and b), which is in line with a previously described interaction between these two proteins (Seeler et al., 1998). The presence of HP1α has also been observed for giant PML bodies in lymphocytes of patients suffering from the ICF syndrome. This PML structure contains a core of cellular satellite DNA, which is present in a hypomethylated and decondensed state in ICF, and therefore suggests a role of PML-NBs in the establishment of condensed heterochomatin (Luciani et al., 2006). However, while HP1 proteins localize to the center of the PML body and are surrounded by concentric layers of other PML-NB components, all proteins detected at HCMV-induced PML cages colocalize at the rim thus suggesting subtle differences in the architecture of giant PML-NBs (Figures 2a and 6a). Several further reports on the formation of enlarged PML-NBs can be found in literature, for instance as alternative lengthening of telomers (ALT)-associated PML-NBs in telomerase-negative tumor cells, in cells quiescently infected with HSV-1, and during infection with BK virus or Merkel cell polyomavirus (Everett et al., 2007; Jiang et al., 2011; Yeager et al., 1999; Neumann et al., 2016). The molecular basis for their formation, however, is far from being understood. Since during HCMV infection, the immediate-early protein IE1 functions as inhibitor of interferon (IFN) signaling, we hypothesized that the drastic enlargement of PML-NBs after infection with IE1-deficient HCMV results from IFN-mediated upregulation of PML-NB proteins (Paulus et al., 2006). This would also fit to the observation that PML cages preferentially appear after infection with high doses of IE1-deficient HCMV (Figure 3a–c). Indeed, block of the IFN pathway reduces the formation of PML cages suggesting an important role of IFN in the development of these structures and corroborating the previous finding that IFN signaling enhances the antiviral activity of restriction factors (Figure 4a–c, Sakuma et al., 2007). However, this effect is not just due to increased protein amounts, as overexpression of PML (Figure 2c) or stimulation of PML-NB protein expression by IFN treatment (Figure 4d–f) is not sufficient to induce PML cages. Our data reveal that induction of a DNA damage response is required as an additional stimulus in HFF cells (Figure 4g and h). While most studies that implicate PML-NBs in the DNA damage response have focused on acute cellular DNA damage, very recent data postulate a role in the repair of persistent DNA damage and even show an association of circular PML structures with such DNA lesions (Imrichova et al., 2019; Vancurova et al., 2019). These PML structures occur after prolonged treatment with DNA damage-inducing agents (Imrichova et al., 2019), which fits to our observations (Figure 4h) and also to the slow kinetics of PML cages biogenesis in HCMVΔIE1-infected cells (Figure 1e). Therefore, it appears likely that PML cages form as a response to continuous interferon and DNA damage signaling during HCMVΔIE1 infection, initiated by incoming viral DNA and enhanced by viral DNA replication, leading to a further enlargement of PML spheres (Figure 3f). The requirement of DNA damage signaling for formation of PML cages is supported by our observations that PML cages exhibit a strong association with markers of DNA damage (Figure 4i, j) and treatment of HCMVΔIE1 infected cells with an ATM inhibitor significantly reduces the number of giant PML-NBs (Figure 4k, l). Of note, IFN signaling was also found to be upregulated in an animal model of ICF syndrome suggesting that dual signaling via the interferon and DNA damage response may constitute a common mechanism whereby entrapment by giant PML nuclear bodies is triggered (Rajshekar et al., 2018).

As a major finding, our data provide evidence that PML can enclose both input viral genomes and newly assembled nuclear capsids, which are distinguishable activities occurring at different times of HCMV infection. To study the association of PML cages with incoming HCMV genomes, we made use of alkyne-modified deoxynucleosides that allow DNA visualization without denaturation and have been successfully utilized to detect adenovirus, vaccinia virus, and herpesvirus DNA (Wang et al., 2013). Concerning sensitivity and impact on virus growth, we found that EdC is most suitable for generation of labeled HCMV (Figure 5—figure supplements 1 and 2a), although EdU and F-ara-EdU have been reported to be incorporated with higher efficiency in short-pulse experiments or being more sensitive for detection of cellular DNA, respectively (Manska et al., 2020; Neef and Luedtke, 2011). EdC labeling efficiency of HCMV DNA was determined by costaining of vDNA with tegument protein pp150, serving as a proxy for viral capsids, and was calculated to reach at least 70% (Figure 5—figure supplement 2c, d). Visualization of invading HCMV and HCMVΔIE1 genomes at immediate-early times of infection revealed an entrapment of viral DNA (vDNA) in slightly enlarged PML-NBs (Figure 5a–e), which is highly efficient in cell nuclei containing only one HCMV genome but significantly reduced with increasing number of viral genomes (Figure 5a, c). Thus, our data provide an explanation for the fact that PML-NB-based restriction of HCMV gene expression can only be observed under low MOI conditions and underline the importance of IE1 for initiation of lytic replication at low MOI (Tavalai et al., 2006). A similar MOI-dependent envelopment of viral genomes has recently been described for HSV-1 DNA upon nuclear entry (Alandijany et al., 2018). Compared to our data on HCMVΔIE1, there appears to be a higher degree of PML-vDNA colocalization indicating a more efficient genome entrapment. However, in contrast to HSV-1, genome entrapment by PML-NBs may already be antagonized prior to de novo viral gene expression by the HCMV tegument protein pp71, which has been shown to disperse ATRX and induce degradation of Daxx. In fact, this scenario is supported by a reduced frequency of PML colocalization with HSV-1 genomes in ATRX-depleted cells (Alandijany et al., 2018). Notably, HCMVΔIE1 input genomes remain associated with PML-NBs and can still be detected in a condensed state at the inner rim of PML cages at 3 days after low multiplicity infection (MOI = 0.1) (Figure 5f). Although PML cages cannot completely silence HCMV transcription at higher MOIs (MOI = 1), as suggested by genome decondensation and IE2 expression (Figure 5f), our data demonstrate a repressive activity because PML disruption under these conditions results in full transcriptional recovery and promotes viral genome replication (Figure 6c–e).

As an independent antiviral activity, PML cages are positioned next to viral replication centers and entrap newly assembled viral capsids. By correlative light and electron microscopy (TEM and FIB-SEM tomography), we identify PML cages as circular clusters of viral capsids that are enveloped by PML fibers and occur after high MOI infection with HCMVΔIE1 (Figure 7c–e; Figure 7—figure supplement 3), but not wild-type HCMV (Figure 7—figure supplement 2S). This gives ground to assume that, after initial disruption of PML-NBs, IE1 remains bound to PML in order to antagonize capsid entrapment at late stages of HCMV infection and may explain the metabolic stability of the immediate-early protein (Scherer et al., 2016). The question remains of how HCMV genomes and capsids are sensed and entrapped by PML cages. The fact that PML cages form in absence of viral DNA replication implies a formation or incorporation of viral capsids in pre-assembled PML structures rather than an active envelopment of nuclear capsids by PML (Figure 3e, f). A similar sequestration of viral capsids by PML cages has been found in cells infected with varicella-zoster virus (VZV) by immuno-electron microscopy (Reichelt et al., 2011). However, a unique C-terminal domain in PML isoform IV is required for both VZV capsid binding and antiviral activity, whereas our data show an isoform-independent formation of PML cages (Figure 2d). This suggests a requirement of the common N-terminal TRIM region for HCMV restriction and correlates with a previously demonstrated anti-HCMV activity of the shortest, nuclear PML isoform VI (Tavalai et al., 2006).

Taken together, we show a multilayered antiviral role of PML-NBs during HCMV infection, which is based on an entrapment mechanism likely leading to a more efficient inhibition or immobilization of viral components. Since the restriction activity of PML at immediate-early times is overcome by high virus doses, entrapment of nuclear capsids may have evolved as an additional line of defense in late infected cells, however, both antiviral strategies are efficiently antagonized by IE1 during wild-type HCMV infection. With regard to previous reports of sequestration events during HSV-1 and VZV infection, we conclude that entrapment of viral components by PML-NBs serves as general mechanism to inhibit herpesviral infections.

All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1, 3, 4, 5, 6 and Figure 5-figure supplement 2.

1. 1. Alandijany T
   2. Roberts APE
   3. Conn KL
   4. Loney C
   5. McFarlane S
   6. Orr A
   7. 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 Pathogens 14:e1006769.

   https://doi.org/10.1371/journal.ppat.1006769

   • PubMed
   • Google Scholar
2. 1. Andreoni M
   2. Faircloth M
   3. Vugler L
   4. Britt WJ

   (1989) A rapid microneutralization assay for the measurement of neutralizing antibody reactive with human cytomegalovirus

   Journal of Virological Methods 23:157–167.

   https://doi.org/10.1016/0166-0934(89)90129-8

   • PubMed
   • Google Scholar
3. 1. Bernardi R
   2. Pandolfi PP

   (2007) Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies

   Nature Reviews. Molecular Cell Biology 8:1006–1016.

   https://doi.org/10.1038/nrm2277

   • PubMed
   • Google Scholar
4. 1. Boutell C
   2. Cuchet-Lourenço D
   3. Vanni E
   4. Orr A
   5. Glass M
   6. McFarlane S
   7. Everett RD

   (2011) A viral ubiquitin ligase has substrate preferential SUMO targeted ubiquitin ligase activity that counteracts intrinsic antiviral defence

   PLOS Pathogens 7:e1002245.

   https://doi.org/10.1371/journal.ppat.1002245

   • PubMed
   • Google Scholar
5. 1. Catez F
   2. Picard C
   3. Held K
   4. Gross S
   5. Rousseau A
   6. Theil D
   7. Sawtell N
   8. Labetoulle M
   9. 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 Pathogens 8:e1002852.

   https://doi.org/10.1371/journal.ppat.1002852

   • PubMed
   • Google Scholar
6. 1. Chee AV
   2. Lopez P
   3. Pandolfi PP
   4. Roizman B

   (2003) Promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects

   Journal of Virology 77:7101–7105.

   https://doi.org/10.1128/jvi.77.12.7101-7105.2003

   • PubMed
   • Google Scholar
7. 1. Chelbi-Alix MK
   2. Pelicano L
   3. Quignon F
   4. Koken MH
   5. Venturini L
   6. Stadler M
   7. Pavlovic J
   8. Degos L
   9. de Thé H

   (1995)

   Induction of the PML protein by interferons in normal and APL cells

   Leukemia 9:2027–2033.

   • PubMed
   • Google Scholar
8. 1. Cuchet D
   2. Sykes A
   3. Nicolas A
   4. Orr A
   5. Murray J
   6. Sirma H
   7. Heeren J
   8. Bartelt A
   9. Everett RD

   (2011) PML isoforms I and II participate in PML-dependent restriction of HSV-1 replication

   Journal of Cell Science 124:280–291.

   https://doi.org/10.1242/jcs.075390

   • PubMed
   • Google Scholar
9. 1. Datsenko KA
   2. Wanner BL

   (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

   PNAS 97:6640–6645.

   https://doi.org/10.1073/pnas.120163297

   • PubMed
   • Google Scholar
10. 1. Day PM
    2. Baker CC
    3. Lowy DR
    4. Schiller JT

    (2004) Establishment of papillomavirus infection is enhanced by promyelocytic leukemia protein (PML) expression

    PNAS 101:14252–14257.

    https://doi.org/10.1073/pnas.0404229101

    • PubMed
    • Google Scholar
11. 1. El Asmi F
    2. Maroui MA
    3. Dutrieux J
    4. Blondel D
    5. Nisole S
    6. Chelbi-Alix MK

    (2014) Implication of PMLIV in Both Intrinsic and Innate Immunity

    PLOS Pathogens 10:e1003975.

    https://doi.org/10.1371/journal.ppat.1003975

    • PubMed
    • Google Scholar
12. 1. Eskiw CH
    2. Dellaire G
    3. Mymryk JS
    4. Bazett-Jones DP

    (2003) Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly

    Journal of Cell Science 116:4455–4466.

    https://doi.org/10.1242/jcs.00758

    • PubMed
    • Google Scholar
13. 1. Everett RD
    2. Chelbi-Alix MK

    (2007) PML and PML nuclear bodies: implications in antiviral defence

    Biochimie 89:819–830.

    https://doi.org/10.1016/j.biochi.2007.01.004

    • PubMed
    • Google Scholar
14. 1. Everett RD
    2. Murray J
    3. Orr A
    4. Preston CM

    (2007) Herpes simplex virus type 1 genomes are associated with ND10 nuclear substructures in quiescently infected human fibroblasts

    Journal of Virology 81:10991–11004.

    https://doi.org/10.1128/JVI.00705-07

    • PubMed
    • Google Scholar
15. 1. Greaves RF
    2. Mocarski ES

    (1998) Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant

    Journal of Virology 72:366–379.

    https://doi.org/10.1128/JVI.72.1.366-379.1998

    • PubMed
    • Google Scholar
16. 1. Grimwade D
    2. Solomon E

    (1997) Characterisation of the PML/RAR alpha rearrangement associated with t(15;17) acute promyelocytic leukaemia

    Current Topics in Microbiology and Immunology 220:81–112.

    https://doi.org/10.1007/978-3-642-60479-9_6

    • PubMed
    • Google Scholar
17. 1. Grobelny JV
    2. Godwin AK
    3. Broccoli D

    (2000) ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle

    Journal of Cell Science 113 Pt 24:4577–4585.

    https://doi.org/10.1242/jcs.113.24.4577

    • PubMed
    • Google Scholar
18. 1. Hofmann H
    2. Flöss S
    3. Stamminger T

    (2000) Covalent modification of the transactivator protein IE2-p86 of human cytomegalovirus by conjugation to the ubiquitin-homologous proteins SUMO-1 and hSMT3b

    Journal of Virology 74:2510–2524.

    https://doi.org/10.1128/jvi.74.6.2510-2524.2000

    • PubMed
    • Google Scholar
19. 1. Imrichova T
    2. Hubackova S
    3. Kucerova A
    4. Kosla J
    5. Bartek J
    6. Hodny Z
    7. Vasicova P

    (2019) Dynamic PML protein nucleolar associations with persistent DNA damage lesions in response to nucleolar stress and senescence-inducing stimuli

    Aging 11:7206–7235.

    https://doi.org/10.18632/aging.102248

    • PubMed
    • Google Scholar
20. 1. Ishov AM
    2. Maul GG

    (1996) The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition

    The Journal of Cell Biology 134:815–826.

    https://doi.org/10.1083/jcb.134.4.815

    • PubMed
    • Google Scholar
21. 1. Ishov AM
    2. Stenberg RM
    3. Maul GG

    (1997) Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment

    The Journal of Cell Biology 138:5–16.

    https://doi.org/10.1083/jcb.138.1.5

    • PubMed
    • Google Scholar
22. 1. Jiang M
    2. Entezami P
    3. Gamez M
    4. Stamminger T
    5. Imperiale MJ

    (2011) Functional reorganization of promyelocytic leukemia nuclear bodies during BK virus infection

    MBio 2:e00281.

    https://doi.org/10.1128/mBio.00281-11

    • PubMed
    • Google Scholar
23. 1. Kim YE
    2. Ahn JH

    (2015) Positive role of promyelocytic leukemia protein in type I interferon response and its regulation by human cytomegalovirus

    PLOS Pathogens 11:e1004785.

    https://doi.org/10.1371/journal.ppat.1004785

    • PubMed
    • Google Scholar
24. 1. Komatsu T
    2. Nagata K
    3. Wodrich H

    (2016) An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies

    Journal of Virology 90:1657–1667.

    https://doi.org/10.1128/JVI.02545-15

    • PubMed
    • Google Scholar
25. 1. Kremer JR
    2. Mastronarde DN
    3. McIntosh JR

    (1996) Computer visualization of three-dimensional image data using IMOD

    Journal of Structural Biology 116:71–76.

    https://doi.org/10.1006/jsbi.1996.0013

    • PubMed
    • Google Scholar
26. 1. Lavau C
    2. Marchio A
    3. Fagioli M
    4. Jansen J
    5. Falini B
    6. Lebon P
    7. Grosveld F
    8. Pandolfi PP
    9. Pelicci PG
    10. Dejean A

    (1995)

    The acute promyelocytic leukaemia-associated PML gene is induced by interferon

    Oncogene 11:871–876.

    • PubMed
    • Google Scholar
27. 1. Li Z
    2. Zhao D
    3. Xiang B
    4. Li H

    (2017) Structural and biochemical characterization of DAXX-ATRX interaction

    Protein & Cell 8:762–766.

    https://doi.org/10.1007/s13238-017-0463-x

    • PubMed
    • Google Scholar
28. 1. Luciani JJ
    2. Depetris D
    3. Usson Y
    4. Metzler-Guillemain C
    5. Mignon-Ravix C
    6. Mitchell MJ
    7. Megarbane A
    8. Sarda P
    9. Sirma H
    10. Moncla A
    11. Feunteun J
    12. Mattei M-G

    (2006) PML nuclear bodies are highly organised DNA-protein structures with a function in heterochromatin remodelling at the G2 phase

    Journal of Cell Science 119:2518–2531.

    https://doi.org/10.1242/jcs.02965

    • PubMed
    • Google Scholar
29. 1. Lukashchuk V
    2. McFarlane S
    3. Everett RD
    4. Preston CM

    (2008) Human cytomegalovirus protein pp71 displaces the chromatin-associated factor ATRX from nuclear domain 10 at early stages of infection

    Journal of Virology 82:12543–12554.

    https://doi.org/10.1128/JVI.01215-08

    • PubMed
    • Google Scholar
30. 1. Manska S
    2. Octaviano R
    3. Rossetto CC

    (2020) 5-Ethynyl-2'-deoxycytidine and 5-ethynyl-2'-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses

    The Journal of Biological Chemistry 295:5871–5890.

    https://doi.org/10.1074/jbc.RA119.012378

    • PubMed
    • Google Scholar
31. 1. Meerbrey KL
    2. Hu G
    3. Kessler JD
    4. Roarty K
    5. Li MZ
    6. Fang JE
    7. Herschkowitz JI
    8. Burrows AE
    9. Ciccia A
    10. Sun T
    11. Schmitt EM
    12. Bernardi RJ
    13. Fu X
    14. Bland CS
    15. Cooper TA
    16. Schiff R
    17. Rosen JM
    18. Westbrook TF
    19. Elledge SJ

    (2011) The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

    PNAS 108:3665–3670.

    https://doi.org/10.1073/pnas.1019736108

    • PubMed
    • Google Scholar
32. 1. Neef AB
    2. Luedtke NW

    (2011) Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides

    PNAS 108:20404–20409.

    https://doi.org/10.1073/pnas.1101126108

    • PubMed
    • Google Scholar
33. 1. Neumann F
    2. Czech-Sioli M
    3. Dobner T
    4. Grundhoff A
    5. Schreiner S
    6. Fischer N

    (2016) Replication of Merkel cell polyomavirus induces reorganization of promyelocytic leukemia nuclear bodies

    The Journal of General Virology 97:2926–2938.

    https://doi.org/10.1099/jgv.0.000593

    • PubMed
    • Google Scholar
34. 1. Paulus C
    2. Krauss S
    3. Nevels M

    (2006) A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling

    PNAS 103:3840–3845.

    https://doi.org/10.1073/pnas.0600007103

    • PubMed
    • Google Scholar
35. 1. Rajshekar S
    2. Yao J
    3. Arnold PK
    4. Payne SG
    5. Zhang Y
    6. Bowman TV
    7. Schmitz RJ
    8. Edwards JR
    9. Goll M

    (2018) Pericentromeric hypomethylation elicits an interferon response in an animal model of ICF syndrome

    eLife 7:e39658.

    https://doi.org/10.7554/eLife.39658

    • PubMed
    • Google Scholar
36. 1. Regad T
    2. Saib A
    3. Lallemand-Breitenbach V
    4. Pandolfi PP
    5. de Thé H
    6. Chelbi-Alix MK

    (2001) PML mediates the interferon-induced antiviral state against a complex retrovirus via its association with the viral transactivator

    The EMBO Journal 20:3495–3505.

    https://doi.org/10.1093/emboj/20.13.3495

    • PubMed
    • Google Scholar
37. 1. Reichelt M
    2. Wang L
    3. Sommer M
    4. Perrino J
    5. Nour AM
    6. Sen N
    7. Baiker A
    8. Zerboni L
    9. Arvin AM

    (2011) Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus

    PLOS Pathogens 7:e1001266.

    https://doi.org/10.1371/journal.ppat.1001266

    • PubMed
    • Google Scholar
38. 1. Reichelt M
    2. Joubert L
    3. Perrino J
    4. Koh AL
    5. Phanwar I
    6. 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 Pathogens 8:e1002740.

    https://doi.org/10.1371/journal.ppat.1002740

    • PubMed
    • Google Scholar
39. 1. Reuter N
    2. Reichel A
    3. Stilp AC
    4. Scherer M
    5. Stamminger T

    (2018) SUMOylation of IE2p86 is required for efficient autorepression of the human cytomegalovirus major immediate-early promoter

    The Journal of General Virology 99:369–378.

    https://doi.org/10.1099/jgv.0.001021

    • PubMed
    • Google Scholar
40. 1. Saffert RT
    2. Kalejta RF

    (2006) Inactivating a cellular intrinsic immune defense mediated by Daxx is the mechanism through which the human cytomegalovirus pp71 protein stimulates viral immediate-early gene expression

    Journal of Virology 80:3863–3871.

    https://doi.org/10.1128/JVI.80.8.3863-3871.2006

    • PubMed
    • Google Scholar
41. 1. Sakuma R
    2. Mael AA
    3. Ikeda Y

    (2007) Alpha interferon enhances TRIM5alpha-mediated antiviral activities in human and rhesus monkey cells

    Journal of Virology 81:10201–10206.

    https://doi.org/10.1128/JVI.00419-07

    • PubMed
    • Google Scholar
42. 1. Scherer M
    2. Klingl S
    3. Sevvana M
    4. Otto V
    5. Schilling EM
    6. Stump JD
    7. Müller R
    8. Reuter N
    9. Sticht H
    10. Muller YA
    11. Stamminger T

    (2014) Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions

    PLOS Pathogens 10:e1004512.

    https://doi.org/10.1371/journal.ppat.1004512

    • PubMed
    • Google Scholar
43. 1. Scherer M
    2. Otto V
    3. Stump JD
    4. Klingl S
    5. Müller R
    6. Reuter N
    7. Muller YA
    8. Sticht H
    9. Stamminger T

    (2016) Characterization of Recombinant Human Cytomegaloviruses Encoding IE1 Mutants L174P and 1-382 Reveals that Viral Targeting of PML Bodies Perturbs both Intrinsic and Innate Immune Responses

    Journal of Virology 90:1190–1205.

    https://doi.org/10.1128/JVI.01973-15

    • PubMed
    • Google Scholar
44. 1. Schilling E-M
    2. Scherer M
    3. Reuter N
    4. Schweininger J
    5. Muller YA
    6. Stamminger T

    (2017) The Human Cytomegalovirus IE1 Protein Antagonizes PML Nuclear Body-Mediated Intrinsic Immunity via the Inhibition of PML De Novo SUMOylation

    Journal of Virology 91:e02049-16.

    https://doi.org/10.1128/JVI.02049-16

    • PubMed
    • Google Scholar
45. 1. Seeler JS
    2. Marchio A
    3. Sitterlin D
    4. Transy C
    5. Dejean A

    (1998) Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment

    PNAS 95:7316–7321.

    https://doi.org/10.1073/pnas.95.13.7316

    • PubMed
    • Google Scholar
46. 1. Tang J
    2. Wu S
    3. Liu H
    4. Stratt R
    5. Barak OG
    6. Shiekhattar R
    7. Picketts DJ
    8. Yang X

    (2004) A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein

    The Journal of Biological Chemistry 279:20369–20377.

    https://doi.org/10.1074/jbc.M401321200

    • PubMed
    • Google Scholar
47. 1. Tavalai N
    2. Papior P
    3. Rechter S
    4. Leis M
    5. Stamminger T

    (2006) Evidence for a role of the cellular ND10 protein PML in mediating intrinsic immunity against human cytomegalovirus infections

    Journal of Virology 80:8006–8018.

    https://doi.org/10.1128/JVI.00743-06

    • PubMed
    • Google Scholar
48. 1. Tavalai N
    2. Stamminger T

    (2008) New insights into the role of the subnuclear structure ND10 for viral infection

    Biochimica et Biophysica Acta 1783:2207–2221.

    https://doi.org/10.1016/j.bbamcr.2008.08.004

    • PubMed
    • Google Scholar
49. 1. Tavalai N
    2. Stamminger T

    (2009) Interplay between Herpesvirus Infection and Host Defense by PML Nuclear Bodies

    Viruses 1:1240–1264.

    https://doi.org/10.3390/v1031240

    • PubMed
    • Google Scholar
50. 1. Tischer BK
    2. von Einem J
    3. Kaufer B
    4. Osterrieder N

    (2006) Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli

    BioTechniques 40:191–197.

    https://doi.org/10.2144/000112096

    • PubMed
    • Google Scholar
51. 1. Ulbricht T
    2. Alzrigat M
    3. Horch A
    4. Reuter N
    5. von Mikecz A
    6. Steimle V
    7. Schmitt E
    8. Krämer OH
    9. Stamminger T
    10. Hemmerich P

    (2012) PML promotes MHC class II gene expression by stabilizing the class II transactivator

    The Journal of Cell Biology 199:49–63.

    https://doi.org/10.1083/jcb.201112015

    • PubMed
    • Google Scholar
52. 1. Vancurova M
    2. Hanzlikova H
    3. Knoblochova L
    4. Kosla J
    5. Majera D
    6. Mistrik M
    7. Burdova K
    8. Hodny Z
    9. Bartek J

    (2019) PML nuclear bodies are recruited to persistent DNA damage lesions in an RNF168-53BP1 dependent manner and contribute to DNA repair

    DNA Repair 78:114–127.

    https://doi.org/10.1016/j.dnarep.2019.04.001

    • PubMed
    • Google Scholar
53. 1. Villinger C
    2. Gregorius H
    3. Kranz C
    4. Höhn K
    5. Münzberg C
    6. von Wichert G
    7. Mizaikoff B
    8. Wanner G
    9. Walther P

    (2012) FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

    Histochemistry and Cell Biology 138:549–556.

    https://doi.org/10.1007/s00418-012-1020-6

    • PubMed
    • Google Scholar
54. 1. Villinger C
    2. Neusser G
    3. Kranz C
    4. Walther P
    5. Mertens T

    (2015) 3D Analysis of HCMV Induced-Nuclear Membrane Structures by FIB/SEM Tomography: Insight into an Unprecedented Membrane Morphology

    Viruses 7:5686–5704.

    https://doi.org/10.3390/v7112900

    • PubMed
    • Google Scholar
55. 1. Wagenknecht N
    2. Reuter N
    3. Scherer M
    4. Reichel A
    5. Müller R
    6. Stamminger T

    (2015) Contribution of the Major ND10 Proteins PML, hDaxx and Sp100 to the Regulation of Human Cytomegalovirus Latency and Lytic Replication in the Monocytic Cell Line THP-1

    Viruses 7:2884–2907.

    https://doi.org/10.3390/v7062751

    • PubMed
    • Google Scholar
56. 1. Waldo FB
    2. Britt WJ
    3. Tomana M
    4. Julian BA
    5. Mestecky J

    (1989) Non-specific mesangial staining with antibodies against cytomegalovirus in immunoglobulin-A nephropathy

    Lancet (London, England) 8630:129–131.

    https://doi.org/10.1016/s0140-6736(89)91144-6

    • Google Scholar
57. 1. Walther P
    2. Ziegler A

    (2002) Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water

    Journal of Microscopy 208:3–10.

    https://doi.org/10.1046/j.1365-2818.2002.01064.x

    • PubMed
    • Google Scholar
58. 1. Wang IH
    2. Suomalainen M
    3. Andriasyan V
    4. Kilcher S
    5. Mercer J
    6. Neef A
    7. Luedtke NW
    8. Greber UF

    (2013) Tracking viral genomes in host cells at single-molecule resolution

    Cell Host & Microbe 14:468–480.

    https://doi.org/10.1016/j.chom.2013.09.004

    • PubMed
    • Google Scholar
59. 1. Weidtkamp-Peters S
    2. Lenser T
    3. Negorev D
    4. Gerstner N
    5. Hofmann TG
    6. Schwanitz G
    7. Hoischen C
    8. Maul G
    9. Dittrich P
    10. Hemmerich P

    (2008) Dynamics of component exchange at PML nuclear bodies

    Journal of Cell Science 121:2731–2743.

    https://doi.org/10.1242/jcs.031922

    • PubMed
    • Google Scholar
60. 1. Yasuda S
    2. Inoue K
    3. Hirabayashi M
    4. Higashiyama H
    5. Yamamoto Y
    6. Fuyuhiro H
    7. Komure O
    8. Tanaka F
    9. Sobue G
    10. Tsuchiya K
    11. Hamada K
    12. Sasaki H
    13. Takeda K
    14. Ichijo H
    15. Kakizuka A

    (1999) Triggering of neuronal cell death by accumulation of activated SEK1 on nuclear polyglutamine aggregations in PML bodies

    Genes to Cells 4:743–756.

    https://doi.org/10.1046/j.1365-2443.1999.00294.x

    • PubMed
    • Google Scholar
61. 1. Yeager TR
    2. Neumann AA
    3. Englezou A
    4. Huschtscha LI
    5. Noble JR
    6. Reddel RR

    (1999)

    Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body

    Cancer Research 59:4175–4179.

    • PubMed
    • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Päivi Ojala as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Marina Lusic (Reviewer #1).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

All reviewers agreed that your work is highly interesting, but also agreed on the necessity to address the question of DNA damage involvement in the formation of PML cages in the context of HCMV infection. In addition, they all noted that quantifications are necessary for proper interpretation of the data, specifically quantitation of input genomes and statistical population analysis.

Reviewer #1 (Recommendations for the authors):

The formation of giant PML bodies in response to IE1 deficient HCMV infection is well supported by the compelling microscopy data showing the entrapment of viral genomes within these structures. The aspects related to the importance of viral titer are very interesting, as it seems that these structures are efficiently entrapping viral genomes (as well as viral capsids at different time points) only at the lower MOIs.

However, judging from the title, the authors might have aimed at showing that IFN signaling and DNA damage play an important role in formation of these cages, as they can be created in cells even in the absence of viral infection. Although their data do suggest that IFN signaling together with DNA damage response plays a perhaps more important role in the initiation of PML accumulation, these observations were not further exploited and some additional controls could allow for a better understanding of the underlying mechanism.

The authors treat HFF with campthotecin or doxyrubicin (inhibitor of TopoI), but do not show any proof of DNA damage. A staining with γH2AX (or TP53BP1) would be helpful to understand if these bodies are formed in the proximity of DNA damage sites.

Another point that I found would be very interesting to address is the turnover of PML within the CAGE like structures. Are these giant PML bodies susceptible to arsenic trioxide degradation? Arsenic has a well documented role in PML turnover and degradation, and is also used as therapeutic agent in treatment of APL. Moreover, it has also been used in clinical trials for the treatment of HTLV, as well as of HIV-1. It would therefore be very interesting to see if the PML can be degraded by arsenic withing these CAGE like structures, and if this has a relevance for viral reactivation.

Reviewer #2 (Recommendations for the authors):

This reviewer recognises the strength of this submission and its contribution to the field. The points below aim to strengthen the study further to provide greater clarity and context to a non-specialist reader.

1. The authors should demonstrate the percentage of viral genomes labelled in their HCMV.EdC virion stock. Based on analogous data derived from HSV-1 (Alandijani et al., 2018), EdC genome labelling efficiency within any given stock of particles can vary. Thus, there may be a significant population of unlabelled HCMV genomes within infected cells that are undetectable by click chemistry. This population should be quantified and presented as supplemental data, as this population of unlabelled genomes may significantly influence the impact and interpretation of data and statements presented in the manuscript. For example lines 271 to 276, 'However, by far not all PML cages contained HCMV genomes suggesting that their enlargement is not a direct consequence of the entrapment process. Taken together, out data provide evidence that genome entrapment by PML-NBs has evolved as a mechanism to achieve efficient and persistent repression of HCMV. Reorganization from dot-like foci to PML cages, however, cannot be directly linked to vDNA entrapment but seems to result from a more general stimulus'.

This statement holds true if 90-100% of genomes delivered to the nucleus are detectable by click chemistry. However, if the percentage of genomes labelled is {less than or equal to} 90%, then the authors cannot make this conclusion, as vDNA undetectable by click chemistry may contribute to the phenotypes they observe. Similarly, the authors should make attempts to quantify the precise genome load delivered to cells at the indicated multiplicities of infection used (currently derived from FFU titration). HCMV virus preparations are well established to have a high genome to PFU ratios. Accurate quantitation will provide greater context to non-specialists as to what low or high MOI means by providing absolute numbers for the indicated MOI.

2. One of the driving conclusions presented by the authors is the requirement for the DDR pathway to be activated to form PML-cages. While the authors present compelling data for this in the context of drug induced DNA damage on uninfected cells (Figure 4g), the authors fail to make the same connection during HCMV infection. The authors should examine the localization of PML-cages induced during deltaIE1-HCMV infection to that of γ-H2Ax, a marker of DNA damage. If their hypothesis holds true, PML-cages should localize in proximity to sites of cellular chromatin damage, thereby providing supporting evidence to one of the papers major conclusions. Alternatively, the authors should investigate the influence of DDR inhibitors (e.g. ATM, ATR, and DNA-PKcs inhibitors) on the frequency PML-cage formation to provide the supporting evidence required to make this conclusion stand during HCMV infection. Correspondingly, if vDNA replication is not required for PML-cage assembly, when is the DDR pathway activated during HCMV infection to promote PML-cage assembly?

3. The manuscript lacks detailed quantitation in its image analysis. While some experiments are quantified, many of the figures (e.g. figures 1 and 2) only show representative images. This makes it hard for the reader to determine the relative population of cells that induce the assembly of PML-cages within any given population of infected cells. Is this phenotype rare or frequent within infected cells? It is recommended that all immunostaining experiments are quantified for readership clarity over a minimum of three independent biological replicates (see below).

Reviewer #3 (Recommendations for the authors):

A. Major items to be addressed

General:

– The reviewer would suggest, to strengthen the physiological relevance of the formation of the PML cages around HCMV genomes and capsids, to perform, if possible, some of the experiments in bone marrow CD34+ cells and/or THP1 cells (already used by the team as HCMV model of latency in previous studies). This, to reviewer's view, could improve significantly, and beyond the molecular aspects, the understanding of the role of the PML NBs in the control of HCMV infection and their role in the establishment of latency and abortive productive infection through the formation of PML cages.

– l446-448: In the discussion the authors state: "The fact that PML cages form in absence of viral DNA replication implies a formation or incorporation of viral capsids in pre-assembled PML structures rather than an active envelopment of nuclear capsids by PML (Figure 3e, f). » This sentence mingles two aspects of the study: (1) The formation of PML cages around incoming viral genomes; and (2) the formation of PML cages containing viral capsids. To reviewer's opinion the statement, as written, does not consider the phase separation properties of the PML protein (Banani et al., 2016), and for reviews see (Lallemand-Breitenbach and Thé, 2018; Corpet et al., 2020), and the fact that 90% of PML protein is not associated with PML NBs, and has a diffuse nuclear localization (see Lallemand-Breitenbach and Thé, 2010) and/or participating to chromatin dynamics (for a review see Delbarre and Janicki, 2021). The reviewer would suggest to clarify this statement, which is, to reviewer's opinion, misleading on the real capacity of PML NBs to form de novo around genome loci (see Everett and Murray. ND10 Components Relocate to Sites Associated with Herpes Simplex Virus Type 1 Nucleoprotein Complexes during Virus Infection, JVI, 2005, 10.1128/jvi.79.8.5078-5089.2005), and/or viral capsids. May be the authors should consider the most likely possibility that it could exist different mechanisms for the formation of PML cages entrapping incoming viral genomes, and PML cages entrapping capsids.

References

Banani SF, Rice AM, Peeples WB, Lin Y, Jain S, Parker R and Rosen MK (2016) Compositional Control of Phase-Separated Cellular Bodies. Cell 166: 651-663

Corpet A, Kleijwegt C, Roubille S, Juillard F, Jacquet K, Texier P and LOMONTE P (2020) PML nuclear bodies and chromatin dynamics: catch me if you can! Nucleic Acids Res 389: 251

Delbarre E and Janicki SM (2021) Modulation of H3.3 chromatin assembly by PML: A way to regulate epigenetic inheritance. Bioessays: 2100038

Lallemand-Breitenbach V and Thé H de (2010) PML nuclear bodies. Cold Spring Harbor perspectives in biology 2: a000661

Lallemand-Breitenbach V and Thé H de (2018) PML nuclear bodies: from architecture to function. Curr Opin Cell Biol 52: 154-161

Specific

– The reviewer acknowledges the importance of IFN signaling and DNA damage for the formation of PML cages, caps or forks in non-infected cells. The requirement of IFN signaling for the formation of PML cages in HCMV-infected cells is clearly shown. However, it is not clear in reviewer's mind how DNA damage provoked by the injection of viral genomes in the nucleus is required to lead to the formation of PML cages specifically around the viral genomes. Does the authors infer that viral genomes themselves would sustain damage that would lead to aggregation of PML around those viral genomes? To reviewer's view the requirement of DNA damage for the formation of PML cages specifically around HCMV genome is not definitively proven. The reviewer would suggest to the authors to perform a γ-H2A.X and PML staining together with click chemistry for the visualization of the genomes at different times post infection to see if PML cages-entrapped viral genomes could at a particular stage of the entry in the nucleus be associated with DNA damage. This could then strengthen the discussion about that aspect.

B. Other

Specific

– What is the fate of the capsids that are entrapped in the PML cages? Are they degraded? Do their remain as an abortive, latency-prone, productive infection? Although the reviewer acknowledges the fact that performing such experiments is beyond the scoop of this study, the reviewer would be grateful to the authors to have their feedback on that question. May be this thought could be part of the discussion.

– During infection of cells supporting HCMV latency what is the status of IE1 expression? In other words, does it exist a physiological situation of infection by HCMV where IE1 cannot be produced, and consequently cannot induce the dispersal of PML NBs? This should be discussed.

– Figure 4g: Could the caps and/or forks in non-infected cells be associated to nucleoli or specific chromosome loci?

- Figure 6c,d,e: The impact of IE1 expression on the abrogation of HCMV repression leads to the increase of viral genomes and to the expression of at least UL44. IF data to visualize UL44 as a marker of the viral replication compartments and/or MCP for capsids in IE1 expressing cells should be provided in addition to the quantification data.

– Figure 6 f and g – The low resolution of the pdf combined to the size of the images make it difficult to conclude anything on the disruption of the APB by IE1. TRF1 is hardly visible in figure 6f. In addition the reviewer would recommend to provide a TRF1 and IE1 staining in figure 6g in addition to the PML/IE1. Indeed, APB are formed by the grouping of several telomeres in the APBs visualized by IF by larger spots of TRF1 (Jiang et al., 2007). It would be interesting to see if those larger spots disappear in IE1-expressing cells.

Reference

Jiang W-Q, Zhong Z-H, Henson JD and Reddel RR (2007) Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference. Oncogene 26: 4635-4647

– The absence of Sp100 does not impact on the formation of the PLM cages but prevent the recruitment of HP1 in those structures. The presence of HP1 in the PML cages could suggest an interaction of HP1 with viral chromatin with nucleosomes containing histone H3 modified on lysine K9 by tri-methylation, for which HP1 is a reader. Could the authors comment on that aspect? Specifically regarding the de novo formation of PML cages around viral genomes by opposition to the relocation of pre-existing PML NBs on incoming viral genomes.

– Although the entrapment of capsids in PML cages is well investigated the question remains to know if the capsids could contain viral genomes or not.

Reviewer #1 (Recommendations for the authors):

The formation of giant PML bodies in response to IE1 deficient HCMV infection is well supported by the compelling microscopy data showing the entrapment of viral genomes within these structures. The aspects related to the importance of viral titer are very interesting, as it seems that these structures are efficiently entrapping viral genomes (as well as viral capsids at different time points) only at the lower MOIs.

However, judging from the title, the authors might have aimed at showing that IFN signaling and DNA damage play an important role in formation of these cages, as they can be created in cells even in the absence of viral infection. Although their data do suggest that IFN signaling together with DNA damage response plays a perhaps more important role in the initiation of PML accumulation, these observations were not further exploited and some additional controls could allow for a better understanding of the underlying mechanism.

The authors treat HFF with campthotecin or doxyrubicin (inhibitor of TopoI), but do not show any proof of DNA damage. A staining with γH2AX (or TP53BP1) would be helpful to understand if these bodies are formed in the proximity of DNA damage sites.

We have performed the suggested γH2AX staining experiment (see Figure 4i, j): it shows that approximately 80% of all PML ring structures exhibit a colocalization with γH2AX signals after treatment of cells with doxorubicin demonstrating a strong association with DNA damage markers.

Another point that I found would be very interesting to address is the turnover of PML within the CAGE like structures. Are these giant PML bodies susceptible to arsenic trioxide degradation? Arsenic has a well documented role in PML turnover and degradation, and is also used as therapeutic agent in treatment of APL. Moreover, it has also been used in clinical trials for the treatment of HTLV, as well as of HIV-1. It would therefore be very interesting to see if the PML can be degraded by arsenic withing these CAGE like structures, and if this has a relevance for viral reactivation.

We have performed the proposed experiment (see Author response image 1): we found that treatment of HCMVΔhIE1 infected cells with arsenic trioxide induces a general degradation of PML. This affected both PML cages and normal PML-NBs structures. Therefore, be believe that metabolization of PML cages after arsenic trioxide treatment occurs via RNF4. We did not include these data in the manuscript because it does not help to differentiate between effects mediated by PML cages and normal PML-NB structures.

Author response image 1

Download asset Open asset

Reviewer #2 (Recommendations for the authors):

This reviewer recognises the strength of this submission and its contribution to the field. The points below aim to strengthen the study further to provide greater clarity and context to a non-specialist reader.

1. The authors should demonstrate the percentage of viral genomes labelled in their HCMV.EdC virion stock. Based on analogous data derived from HSV-1 (Alandijani et al., 2018), EdC genome labelling efficiency within any given stock of particles can vary. Thus, there may be a significant population of unlabelled HCMV genomes within infected cells that are undetectable by click chemistry. This population should be quantified and presented as supplemental data, as this population of unlabelled genomes may significantly influence the impact and interpretation of data and statements presented in the manuscript. For example lines 271 to 276, 'However, by far not all PML cages contained HCMV genomes suggesting that their enlargement is not a direct consequence of the entrapment process. Taken together, out data provide evidence that genome entrapment by PML-NBs has evolved as a mechanism to achieve efficient and persistent repression of HCMV. Reorganization from dot-like foci to PML cages, however, cannot be directly linked to vDNA entrapment but seems to result from a more general stimulus'.

This statement holds true if 90-100% of genomes delivered to the nucleus are detectable by click chemistry. However, if the percentage of genomes labelled is {less than or equal to} 90%, then the authors cannot make this conclusion, as vDNA undetectable by click chemistry may contribute to the phenotypes they observe. Similarly, the authors should make attempts to quantify the precise genome load delivered to cells at the indicated multiplicities of infection used (currently derived from FFU titration). HCMV virus preparations are well established to have a high genome to PFU ratios. Accurate quantitation will provide greater context to non-specialists as to what low or high MOI means by providing absolute numbers for the indicated MOI.

We agree with reviewer #2 that the presence of a significant population of unlabeled viral genomes that are undetectable by click chemistry would affect the interpretation of our results. We tried several times to quantify the labeling efficiency of HCMV genomes as described by Alandijany et al., 2018, for herpes simplex virus. However, it turned out that, most probably due to differences in the stability between HCMV and HSV-1 capsids, partial denaturation with 2 M GuHCl does not lead to an ordered release of HCMV vDNA. Thus, we were not able to use this technique for the assessment of vDNA labelling efficiency. Alternatively, we used a method as developed by Christian Sinzger and colleagues (Sinzger et al., J.Gen.Virol. 2007): this method uses pp150 staining as a proxy for capsid localization at the nuclear rim correlating to capsids that have released viral DNA into the nucleus. Pp150 signals located at cell nuclei as well as viral DNA signals in the same nuclei were counted in order to calculate the EdU/EdC labelling efficiency (see Figure 5—figure supplement 2 c, d). From this, we conclude that the EdC labelling efficiency of HCMV DNA is at least 70% (Figure 5—figure supplement 2 d). Although we agree with reviewer #1 that this may lead to a slight underestimation of the number of PML cages associated with vDNA, we still think that our assumption that enlargement is not a direct consequence of the entrapment process holds true due to the facts that vDNA signals were found in both small and large PML foci and, in addition, not all of the large PML cages contained HCMV genomes (see lines 283 – 285).

Furthermore, we agree with reviewer #1 that HCMV virus preparations have a high genome to PFU ratio. This is even aggravated in nucleoside labelling experiments of viral genomes due the necessary purification procedure. Figure 5—figure supplement 2, panel b, shows the quantification of viral genomes in supernatants and infected cells (nuclei and cytoplasm) in relation to immediate-early units (IEUs). According to this quantification, the ratio between intracellular genome equivalents and IEUs was 55. However, the exact quantification of genomes delivered to nuclei is difficult since many genomes as detected by qPCR in infected cells (see Figure 5—figure supplement 2) do not reach the nucleus. By counting vDNA signals of cells infected at an MOI of 0.1 we calculated that this results, on average, in 1.2 viral genomes/cell (Figure 5—figure supplement 2, panel c). However, we anticipate that this value may not only depend on the used virus stock but also on cellular factors (for instance, on the passage number of primary fibroblasts; e.g. HFFs stably expressing individual PML isoforms may vary in their passage number due to the necessary selection procedure). For this reason, we think that accurate quantification is only valid if done for a specific experimental condition.

2. One of the driving conclusions presented by the authors is the requirement for the DDR pathway to be activated to form PML-cages. While the authors present compelling data for this in the context of drug induced DNA damage on uninfected cells (Figure 4g), the authors fail to make the same connection during HCMV infection. The authors should examine the localization of PML-cages induced during deltaIE1-HCMV infection to that of γ-H2Ax, a marker of DNA damage. If their hypothesis holds true, PML-cages should localize in proximity to sites of cellular chromatin damage, thereby providing supporting evidence to one of the papers major conclusions. Alternatively, the authors should investigate the influence of DDR inhibitors (e.g. ATM, ATR, and DNA-PKcs inhibitors) on the frequency PML-cage formation to provide the supporting evidence required to make this conclusion stand during HCMV infection. Correspondingly, if vDNA replication is not required for PML-cage assembly, when is the DDR pathway activated during HCMV infection to promote PML-cage assembly?

Our response:

We have performed the suggested experiments:

– Figures 4i and j show that PML cages co-localize with γH2AX in IFN/Dox-treated non-infected HFFs;

– Figures 5g and h demonstrate an association of entrapped vDNA with DNA damage sites after infection with HCMVΔhIE1;

– Figures 4k and l show that treatment of HCMVΔhIE1-infected cells with the ATM inhibitor Ku55933 results in a statistically significant reduction of enlarged PML cages. Since this could be detected both after high (viral DNA replication is initiated) and low MOI infection (no viral DNA replication occurs), we conclude that incoming viral genomes are sufficient to trigger DNA damage signaling.

3. The manuscript lacks detailed quantitation in its image analysis. While some experiments are quantified, many of the figures (e.g. figures 1 and 2) only show representative images. This makes it hard for the reader to determine the relative population of cells that induce the assembly of PML-cages within any given population of infected cells. Is this phenotype rare or frequent within infected cells? It is recommended that all immunostaining experiments are quantified for readership clarity over a minimum of three independent biological replicates (see below).

We have performed detailed quantifications over at least three independent biological replicates for the experiments of Figures: 1 (see panel b); 3 (see panels b and f); 4 (see panels f, h, j, k and l); 5 (see panels a, d and h); 6 (see panels d and e), Figure 5—figure supplement 2.

Reviewer #3 (Recommendations for the authors):

A. Major items to be addressed

General:

– The reviewer would suggest, to strengthen the physiological relevance of the formation of the PML cages around HCMV genomes and capsids, to perform, if possible, some of the experiments in bone marrow CD34+ cells and/or THP1 cells (already used by the team as HCMV model of latency in previous studies). This, to reviewer's view, could improve significantly, and beyond the molecular aspects, the understanding of the role of the PML NBs in the control of HCMV infection and their role in the establishment of latency and abortive productive infection through the formation of PML cages.

As already stated above, we have previously shown that THP1 cells with an shRNA instituted knockdown of PML do not exhibit a defect concerning the establishment of HCMV latency (Wagenknecht et al., Viruses 2015). Thus, we assume that cellular repressors different from PML-NBs are decisive for HCMV latency establishment.

– l446-448: In the discussion the authors state: "The fact that PML cages form in absence of viral DNA replication implies a formation or incorporation of viral capsids in pre-assembled PML structures rather than an active envelopment of nuclear capsids by PML (Figure 3e, f). » This sentence mingles two aspects of the study : (1) The formation of PML cages around incoming viral genomes; and (2) the formation of PML cages containing viral capsids. To reviewer's opinion the statement, as written, does not consider the phase separation properties of the PML protein (Banani et al., 2016), and for reviews see (Lallemand-Breitenbach and Thé, 2018; Corpet et al., 2020), and the fact that 90% of PML protein is not associated with PML NBs, and has a diffuse nuclear localization (see Lallemand-Breitenbach and Thé, 2010) and/or participating to chromatin dynamics (for a review see Delbarre and Janicki, 2021). The reviewer would suggest to clarify this statement, which is, to reviewer's opinion, misleading on the real capacity of PML NBs to form de novo around genome loci (see Everett and Murray. ND10 Components Relocate to Sites Associated with Herpes Simplex Virus Type 1 Nucleoprotein Complexes during Virus Infection, JVI, 2005, 10.1128/jvi.79.8.5078-5089.2005), and/or viral capsids. May be the authors should consider the most likely possibility that it could exist different mechanisms for the formation of PML cages entrapping incoming viral genomes, and PML cages entrapping capsids.

References

Banani SF, Rice AM, Peeples WB, Lin Y, Jain S, Parker R and Rosen MK (2016) Compositional Control of Phase-Separated Cellular Bodies. Cell 166: 651-663

Corpet A, Kleijwegt C, Roubille S, Juillard F, Jacquet K, Texier P and LOMONTE P (2020) PML nuclear bodies and chromatin dynamics: catch me if you can! Nucleic Acids Res 389: 251

Delbarre E and Janicki SM (2021) Modulation of H3.3 chromatin assembly by PML: A way to regulate epigenetic inheritance. Bioessays: 2100038

Lallemand-Breitenbach V and Thé H de (2010) PML nuclear bodies. Cold Spring Harbor perspectives in biology 2: a000661

Lallemand-Breitenbach V and Thé H de (2018) PML nuclear bodies: from architecture to function. Curr Opin Cell Biol 52: 154-161

We are well aware of the fact that PML-NBs can form de novo around genome loci (as published by Everett and Murray in 2015). We do not intend to mingle “formation of PML cages around incoming viral genomes” and “formation of PML cages containing viral capsids”. As defined on page 6 of the manuscript the term PML cages is exclusively reserved for enlarged, ring-like PML structures that develop during the late phase of infection. However, we noticed that in some sentences the term PML cages was used, however, instead, PML-NBs were meant. This has been corrected in the revised manuscript. Entrapment of incoming viral genomes as described by Everett and Murray is due to PML-NBs and not due to PML cages. The statement of the Discussion "The fact that PML cages form in absence of viral DNA replication implies a formation or incorporation of viral capsids in pre-assembled PML structures rather than an active envelopment of nuclear capsids by PML” only refers to enlarged PML ring structures surrounding viral capsids. The statement should indicate that PML cages form in the absence of viral capsids. One potential interpretation is that ring-like PML cages are present in a pre-assembled form and subsequently associate with capsids. The statement does not exclude other mechanisms.

Specific

– The reviewer acknowledges the importance of IFN signaling and DNA damage for the formation of PML cages, caps or forks in non-infected cells. The requirement of IFN signaling for the formation of PML cages in HCMV-infected cells is clearly shown. However, it is not clear in reviewer's mind how DNA damage provoked by the injection of viral genomes in the nucleus is required to lead to the formation of PML cages specifically around the viral genomes. Does the authors infer that viral genomes themselves would sustain damage that would lead to aggregation of PML around those viral genomes? To reviewer's view the requirement of DNA damage for the formation of PML cages specifically around HCMV genome is not definitively proven. The reviewer would suggest to the authors to perform a γ-H2A.X and PML staining together with click chemistry for the visualization of the genomes at different times post infection to see if PML cages-entrapped viral genomes could at a particular stage of the entry in the nucleus be associated with DNA damage. This could then strengthen the discussion about that aspect.

As already stated in our response to issues raised by reviewers #1 and #2, we have performed co-staining of vDNA with γH2AX at 8 and 72 hpi (see Figure 5g and h of revised manuscript). Furthermore, we show that inhibition of DNA damage signalling by the ATM inhibitor Ku 55933 significantly reduces the formation of PML cages (see Figure 4k and l).

B. Other

Specific

– What is the fate of the capsids that are entrapped in the PML cages? Are they degraded? Do their remain as an abortive, latency-prone, productive infection? Although the reviewer acknowledges the fact that performing such experiments is beyond the scoop of this study, the reviewer would be grateful to the authors to have their feedback on that question. May be this thought could be part of the discussion.

We agree with reviewer #3 that a final answer to this question will require further experimentation. However, since we observed that IE1 expression disrupts PML cages and this correlates with an increase of viral genomes and gene expression, we would assume that PML cage disruption might also result in a release of entrapped capsids (see Figure 6d and e).

– During infection of cells supporting HCMV latency what is the status of IE1 expression? In other words, does it exist a physiological situation of infection by HCMV where IE1 cannot be produced, and consequently cannot induce the dispersal of PML NBs? This should be discussed.

Many authors define HCMV latency as a status of transcriptional silencing of the HCMV MIE locus (e.g. reviewed in Dooley and O’Connor, Pathogens 2020). Consequently, IE1 is not expressed during latency. However, since our paper does not address the role of PML-NBs during latency, we do not want to include this in the Discussion.

– Figure 4g: Could the caps and/or forks in non-infected cells be associated to nucleoli or specific chromosome loci?

Since previous publications reported on PML protein nucleolar associations upon DNA damage induced senescence it is possible that at least some of the enlarged PML structures could be located at nucleoli (Imrichova et al., Aging 2019).

- Figure 6c,d,e: The impact of IE1 expression on the abrogation of HCMV repression leads to the increase of viral genomes and to the expression of at least UL44. IF data to visualize UL44 as a marker of the viral replication compartments and/or MCP for capsids in IE1 expressing cells should be provided in addition to the quantification data.

We have now included IF data to visualize UL44 as a marker of viral replication compartments (see Figure 6c).

– Figure 6 f and g – The low resolution of the pdf combined to the size of the

images make it difficult to conclude anything on the disruption of the APB by IE1. TRF1 is hardly visible in figure 6f. In addition the reviewer would recommend to provide a TRF1 and IE1 staining in figure 6g in addition to the PML/IE1. Indeed, APB are formed by the grouping of several telomeres in the APBs visualized by IF by larger spots of TRF1 (Jiang et al., 2007). It would be interesting to see if those larger spots disappear in IE1-expressing cells.

Reference

Jiang W-Q, Zhong Z-H, Henson JD and Reddel RR (2007) Identification of candidate alternative lengthening of telomeres genes by methionine restriction and RNA interference. Oncogene 26: 4635-4647

It is well recognizable in Figure 6g that expression of IE1 induces a complete dispersal of all PML dot-like accumulations. This was true for all cells that were positive for IE1. For that reason, we do not think that a co-staining of TRF1 together with IE1 would give additional insights. We agree that for Figure 6f the signals for TRF1 are hardly visible. Therefore, we exchanged the respective figure (see new Figure 6 f).

– The absence of Sp100 does not impact on the formation of the PLM cages but prevent the recruitment of HP1 in those structures. The presence of HP1 in the PML cages could suggest an interaction of HP1 with viral chromatin with nucleosomes containing histone H3 modified on lysine K9 by tri-methylation, for which HP1 is a reader. Could the authors comment on that aspect? Specifically regarding the de novo formation of PML cages around viral genomes by opposition to the relocation of pre-existing PML NBs on incoming viral genomes.

We would assume that HP1 contributes to HCMV genome repression. Future experiments might verify this by using HP1 knockdown cells in infection experiments. Concerning the question of de novo formation of PML cages versus relocation of pre-exisiting PML-NBs on incoming viral genomes: we already clarified above that Everett and Murray convincingly demonstrated the de novo formation of PML-NBs on incoming viral genomes. PML cages, however, only form late during infection and may represent pre-existing structures to associate with viral capsids.

– Although the entrapment of capsids in PML cages is well investigated the question remains to know if the capsids could contain viral genomes or not.

We agree with reviewer #3 that this is an open question that we plan to address in future experiments.

Author details

1. Myriam Scherer

   Institute of Virology, Ulm University Medical Center, Ulm, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Clarissa Read

   1. Institute of Virology, Ulm University Medical Center, Ulm, Germany
   2. Central Facility for Electron Microscopy, Ulm University, Ulm, Germany

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft

   Competing interests

   No competing interests declared

3. Gregor Neusser

   Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Software

   Competing interests

   No competing interests declared

4. Christine Kranz

   Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

5. Anna K Kuderna

   Institute of Virology, Ulm University Medical Center, Ulm, Germany

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Regina Müller

   Institute of Clinical and Molecular Virology, Friedrich Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

7. Florian Full

   Institute of Clinical and Molecular Virology, Friedrich Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. Sonja Wörz

   Institute of Virology, Ulm University Medical Center, Ulm, Germany

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

9. Anna Reichel

   Institute of Virology, Ulm University Medical Center, Ulm, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Validation, Visualization

   Competing interests

   No competing interests declared

10. Eva-Maria Schilling

    Institute of Virology, Ulm University Medical Center, Ulm, Germany

    Contribution

    Data curation, Investigation, Methodology, Validation

    Competing interests

    No competing interests declared

11. Paul Walther

    Central Facility for Electron Microscopy, Ulm University, Ulm, Germany

    Contribution

    Investigation, Methodology

    Competing interests

    No competing interests declared

12. Thomas Stamminger

    Institute of Virology, Ulm University Medical Center, Ulm, Germany

    Contribution

    Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    thomas.stamminger@uni-ulm.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9878-3119

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