Dual signaling via interferon and DNA damage response elicits entrapment by giant PML nuclear bodies

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.

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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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