Small ubiquitin-related modifier-dependent dynamics of PML NBs shapes the outcome of infection with HSV-1, an alphaherpesvirus of the Herpesviridae family which generally causes oral sores. Nuclear entry of the viral genomic DNA triggers host cells to mount an intrinsic antiviral response. An essential facet of this response is the silencing of the viral gene expression and onset of latency. This latent reservoir of virus signifies an evolutionary achievement of the virus as it bypasses immune clearance and has the potential to get reactivated periodically leading to disease manifestation and viral transmission. Interestingly, SUMO-dependent regulations are important for initiating this latency. The incoming viral genome, which is a linear molecule of DNA, adsorbs a plethora of both pro- and antiviral host cellular factors (Dembowski and DeLuca, 2018). These include the H3.3 histone chaperone complex Daxx/ATRX which further enables subsequent entrapment of the viral DNA (vDNA) within PML NBs. Other PML NB resident proteins, such as PML and Sp100, as well as the nuclear DNA sensing protein IFI16 also deposit onto the viral genome, where they co-localize with ICP4, an immediate early gene that serves as a marker for incoming HSV-1 genomes (Everett, 2015; Diner et al., 2016; Alandijany et al., 2018; Cabral et al., 2018). Interestingly, SUMO modification of PML and SIMs of PML, Sp100 and Daxx are pivotal for the association of vDNA with PML NBs (Cuchet-Lourenço et al., 2011). Engulfment of the vDNA within PML NBs coincides with the repressive epigenetic modification of the histones (H3K9me3, H3K27me3) on vDNA leading to a transcriptionally inactive chromatin environment (Lee et al., 2016; Cabral et al., 2018; Cohen et al., 2018). The importance of SUMO conjugation to PML and PML-associated NB components is further supported by the finding that host proteins, which promote SUMO conjugation to PML and PML-NB associated proteins curtail HSV-1 infection (Boutell et al., 2011; Brown et al., 2016; Conn et al., 2016). For example, PIAS1, which localizes to PML NBs and acts as a SUMO E3 ligase on PML, contributes to HSV-1 restriction (Brown et al., 2016; Li et al., 2020).

During infection with the wild type virus, the PML NB association of vDNA is transient as the HSV-1 protein ICP0 counteracts this quiescence by dispersal of PML NBs. ICP0 is dispensable for the establishment and maintenance of HSV-1 latency but very critical for the productive rejuvenation of the viral genome from latency and further onset of lytic replication (Leib et al., 1989; Cai et al., 1993; Halford and Schaffer, 2001). Infection with an ICP0 null mutant results in a more persistent association between HSV-1 genome and PML NBs, whereas expression of ICP0 triggers dismantling of PML NB associated proteins from vDNA (Everett et al., 2013a; Alandijany et al., 2018; Cabral et al., 2018). This results in a reduction of histone H3 deposition and enhanced acetylation of remaining histone H3 on vDNA to promote viral transcription (Cliffe and Knipe, 2008; Ferenczy et al., 2011; Lee et al., 2016; Cabral et al., 2018). These observations support a model, in which sequestration of the HSV-1 genome in PML NBs induces repression of viral gene expression, while ICP0-mediated destruction of NBs counters this process.

Core to the functions of ICP0 is its ability to act as a viral StUbL, thereby triggering RING finger-dependent proteolytic ubiquitylation of SUMO-conjugated proteins (Figures 3, 4), including PML (Boutell et al., 2003, 2011; Lanfranca et al., 2014). Accordingly, mutants of HSV-1 which lack ICP0 or express a ligase deficient ICP0, have a dramatically lower probability of initiating a productive infection in restrictive cell types (Everett et al., 2004). Reminiscent to the StUbLs RNF4 or RNF111, ICP0 harbors a RING-domain and 7 putative SIM-Like Sequences (SLS). Accordingly, IPC0 possesses biochemical properties which are similar to host cellular StUbLs (Everett et al., 1998; Boutell et al., 2003, 2011; Sloan et al., 2015). The structure of ICP0 SLS4 (residue 362–367) with SUMO has been resolved by NMR, revealing a cooperation between ICP0 phosphorylation domains (FHA [67-pTELF-70] and Phos2) for recognition of the SUMOylated proteins (Boutell et al., 2008; Mostafa et al., 2013; Hembram et al., 2020). Therefore, both the ICP0 RING-finger and phosphorylation are important for the degradation of SUMOylated proteins and successful reactivation of HSV-1 from latency (Mostafa et al., 2011, 2013; Vanni et al., 2012). One specific SIM in the central region of ICP0 is absolutely essential for degradation of an array of SUMOylated host proteins, including PML and Sp100 (Figure 3; Everett et al., 1998, 2009, 2014; Müller and Dejean, 1999).

ICP0 also induces the degradation of ATRX and IFI16 (Figure 3; Jurak et al., 2012; Orzalli et al., 2012, 2016; Diner et al., 2015b), but turnover of ATRX and IFI16 occurs with delayed kinetics relative to that of PML (Jurak et al., 2012; Cuchet-Lourenço et al., 2013), possibly indicating that degradation of host components occurs sequentially as infection progresses (Merkl and Knipe, 2019). This is consistent with the step-wise accumulation of cellular factors on vDNA throughout the initiating cycle of HSV- 1 infection (Dembowski and DeLuca, 2015, Dembowski and Deluca, 2017). Recent studies have identified the histone H3.3 chaperone protein HIRA to restrict HSV-1 infection following the onset of vDNA replication, a host response antagonized by ICP0 through the nuclear dispersal of HIRA (McFarlane et al., 2019). Thus, multiple histone H3.3 chaperone proteins (Daxx/ATRX and HIRA) can restrict the replication of ICP0 null HSV-1 at independent phases of infection (Rodríguez et al., 2020). Notably, ICP0 null mutants exhibit reduced plaque forming efficiency which can be reversed by co-depletion of either PML, Sp100, Daxx or ATRX (Everett et al., 2006, 2008; Lukashchuk and Everett, 2010). Absence of a single component does not affect the recruitment of other factors onto HSV-1 genomes, indicating their independent deposition onto the viral genome (Everett et al., 2006, 2008; Lukashchuk and Everett, 2010). Consistent with this, simultaneous depletion of PML and Sp100 enhances ICP0-null HSV-1 propagation greater than single depletion of each factor (Everett et al., 2008). However, another study reported knockdown of PML to enhance wild type HSV-1 propagation (Diner et al., 2015a). Interestingly, SIM-deficient mutants of PML and Daxx are not recruited to vDNA and are incapable of reproducing the repression of ICP0 null mutant HSV-1 viruses (Cuchet-Lourenço et al., 2011), indicating that SIM-dependent PML NB recruitment to HSV-1 DNA during the initial phase of infection is needed to create a transcriptionally repressive environment. Of note, though Sp100 also contributes to the repression of gene expression from null ICP0 HSV-1 virus (Everett et al., 2008, 2009; Glass and Everett, 2013), direct implications of Sp100 on the epigenetic regulation of viral chromatin or modulating innate immune signaling are yet to be determined.

Apart from PML NB resident proteins, a number of host cellular SUMO-modified proteins have now been identified to be targeted by ICP0 (Everett et al., 1998; Chelbi-Alix and de Thé, 1999; Boutell et al., 2011; Sloan et al., 2015). For many of these targets, however, specificity and relevance to HSV-1 replication have yet to be determined, implicating a scenario where at least some of these proteins may be the subjects of “bystander” degradation. Importantly, upon degradation of MORC3 which is a SUMO-conjugated PML NB client targeted by ICP0, recruitment of other PML NB components PML, Sp100, Daxx on to vDNA is also inhibited, substantiating the importance of these foci in defense against invading pathogens (Sloan et al., 2016).

In addition to disarming PML NB-oriented intrinsic immunity, ICP0 also inactivates innate immune defenses early in the infectious cycle through the degradation of cellular PRRs. Detection of viral nucleic acid by host PRRs can elicit a signal transduction cascade leading to generation of IFNs and mobilization of IFN-dependent downstream signaling to produce hundreds of ISGs with antiviral potency. Interestingly, null ICP0 HSV-1 mutants are hypersensitive to IFN (Leib et al., 1999; Mossman et al., 2000; Härle et al., 2002; Everett et al., 2004), indicating that ICP0 limits the IFN-dependent innate immune response. PML II and IV directly influence the induction of cytokines and ISGs by facilitating the loading of transcription factors (including IRF3, NF-κB, and STAT1) onto cellular gene promoters, possibly explaining the IFN hypersensitivity phenotype of null ICP0 HSV-1 mutants (Chee et al., 2003; El Asmi et al., 2014; Chen et al., 2015; McFarlane et al., 2019). Subsequent studies revealed that ICP0 disarms nuclear PRR by degrading IFI16 which recognizes viral dsDNA and signals via STING-dependent pathway and also by triggering decay of DNA-PKcs (Orzalli et al., 2012, 2015, 2016; Cuchet-Lourenço et al., 2013; Diner et al., 2016; Burleigh et al., 2020). Interestingly, a recent study showed a distinct temporal segregation between the entrapment of HSV-1 DNA by PML NBs upon its nuclear entry and the subsequent deposition of IFI16 leading to ISG expression (Alandijany et al., 2018). While the former contributes to the host cellular intrinsic defense measure by triggering viral latency, the latter is related to the host innate immune response. Moreover, PML NB entrapment of vDNA upon its nuclear entry was shown to be independent of IFI16 deposition onto the viral genome which was shown to require vDNA replication. PRR sensing by IFI16 during null ICP0 HSV-1 infection correlates with IFI16 forming nuclear filaments on vDNA in association with PML following the saturation of PML NBs under high genome loads (Cuchet-Lourenço et al., 2013; Alandijany et al., 2018; Merkl and Knipe, 2019). A close functional relationship between innate and intrinsic immunity is further evidenced when resident PML NB proteins (PML, Daxx and ATRX) have been shown to act cooperatively with IFI16 to restrict null ICP0 HSV-1 gene expression that correlates with repressive histone signatures (H3K9me3 and H3K27me3) on viral chromatin (Everett et al., 2008; Lee et al., 2016; Cabral et al., 2018; Merkl et al., 2018).

Another alphaherpesvirus VZV is the etiological agent of chickenpox and the reactivation disease known as shingles (Gilden et al., 2015). Disruption of PML NBs without the reduction of PML protein stability itself has been evidenced in VZV infected cells both in cell culture based studies and in vivo (Kyratsous and Silverstein, 2009; Reichelt et al., 2011). The viral trigger has been found to be the protein ORF61 which is homologous to HSV-1 ICP0 in having a RING finger and SIMs (Figure 3; Moriuchi et al., 1992; Everett et al., 2010; Wang et al., 2011). Unlike ICP0, however, StUbL activity of ORF61 has not yet been formally determined. Nonetheless, Sp100 is targeted for degradation by ORF61 especially during early hours of infection in a ORF61 RING finger-dependent manner (Figure 3; Kyratsous et al., 2009; Kyratsous and Silverstein, 2009; Walters et al., 2010). The significance of this process on VZV life cycle has not been addressed yet. Interestingly, three SIMs within ORF61 were found to be indispensable for targeting of PML NBs. In response to the ORF61 SIM mutant virus, viral replication in skin was severely hampered, the typical VZV lesions were reduced, and viral spread in the epidermis was limited (Wang et al., 2011), suggesting that the SUMO binding function of ORF61 is critical for overcoming the anti-viral effects of the PML bodies. Of interest, as a part of the dynamic host-virus interactions, the persisting PML NBs in VZV infected cells can also mount a potent anitiviral response by sequestering viral nucleocapsids. Mechanistic studies revealed that PML IV interacts with the VZV capsid protein ORF23 to enable this sequestration and to prevent nuclear egress of viral nucleocapsids, thereby inhibiting formation of infectious virions (Figure 3; Reichelt et al., 2011). However, a direct involvement of host cellular SUMO system in this antiviral defense mechanism has remained elusive.

Human cytomegalovirus is a betaherpesvirus which also has the potency to go into latency or to resume lytic reactivation and proliferation. Importantly, transcription of the viral immediate early (IE) genes is crucial for switching the viral infection cycle toward lytic proliferation. Major HCMV IE genes are transcribed under the control of the major immediate early promoter (MIEP), which is only transactivated by a viral protein residing within the tegument layer of infectious virions.

Dynamic SUMO modifications have been reported to contribute to the regulations of intrinsic and innate immune defenses against infection with HCMV. The initial observation that herpesviral DNA associates with PML NBs upon its nuclear entry led to the hypothesis that these condensates are usurped by cytomegalovirus and other herpesviruses for productive initiation of the viral IE gene expression (Maul et al., 1996; Ishov and Maul, 1996; Ishov et al., 1997). Subsequent investigations revealed that these spatial proximity and entrapment of vDNA within PML NBs during initial hours of infection constitute a major arm of the cellular intrinsic antiviral defense (Saffert and Kalejta, 2006; Tavalai et al., 2006, 2008; Woodhall et al., 2006; Adler et al., 2011). This is enabled by creating a transcriptionally–incompetent chromatin environment on vDNA leading to silencing of viral IE gene expression. Agreeably, knocking down expression of individual PML NB components such as PML, Daxx, and Sp100 in primary human fibroblasts showed a de-repressing effect on HCMV IE gene expression at low multiplicity of infection (Saffert and Kalejta, 2006; Tavalai et al., 2006, 2008; Woodhall et al., 2006; Adler et al., 2011). Moreover, simultaneous depletion of more than one PML NB component has additive effects on the number of IE-positive cells than when single knock down was achieved, suggesting that multiple NB components independently contribute to restriction of HCMV lytic replication (Tavalai et al., 2008; Adler et al., 2011). Interestingly, PML NB components play a crucial role in epigenetic modulations of the MIEP, leading to repression of the viral IE gene expression (Groves et al., 2009; Sinclair, 2010). The contribution of Daxx to trigger epigenetic quiescence of viral gene expression is evident from analyzing the chromatin signatures around the MIEP in Daxx-deficient cells (Woodhall et al., 2006). Daxx has also been shown to recruit HDACs and ATRX to the viral promoter to execute transcriptional repression (Saffert and Kalejta, 2006; Lukashchuk et al., 2008). Not surprisingly, therefore, inhibition of HDACs by trichostatin A or depletion of ATRX had a de-repressing effect on HCMV IE gene expression (Saffert and Kalejta, 2006; Lukashchuk et al., 2008). In addition to Daxx, PML has also been reported to interact with HDACs and histone methyl transferases (HMTs) and Sp100 with heterochromatin protein 1 (HP1) (Seeler et al., 1998; Wu et al., 2001; Di Croce et al., 2002; Wilcox et al., 2005; Carbone et al., 2006). However, implications of such interactions in mediating epigenetic silencing of viral genome are yet to be addressed experimentally. Although some mechanistic details are still unclear, the ensemble of these data indicate that - in analogy to HSV1- the sequestration of the HCMV genome in PML NBs mediates the repression of viral gene expression through epigenetic silencing of the genome.

Importantly, however, HCMV has also developed countermeasures to rejuvenate its life cycle and to maintain perpetuation. There are three effector proteins, the tegument protein pp71, the Latency Unique Natural Antigen (LUNA) protein and the major immediate-early protein IE1, which can affect the integrity of PML NBs (Figure 3). The mechanisms employed, however, are different. After being transported to the nucleus during infection of terminally differentiated cells such as fibroblasts and epithelial cells, pp71 interacts with and triggers proteasomal degradation of Daxx and also displaces ATRX from the PML NB condensates (Figure 3; Hofmann et al., 2002; Saffert and Kalejta, 2006; Lukashchuk et al., 2008). This further relieves the Daxx-mediated transcriptional repression on the viral genome leading to expression of viral IE1 from MIEP and productive viral infection. A pp71-null HCMV virus has a major defect in IE gene expression and productive replication (Bresnahan and Shenk, 2000; Cantrell and Bresnahan, 2006). Following infection of incompletely differentiated myeloid cells such as CD34⁺ hematopoietic progenitor cells (HPCs) and monocytes, pp71 remains trapped in cytoplasmic endosomes (Penkert and Kalejta, 2010, 2013; Lee et al., 2019; Lee and Kalejta, 2019; Kalejta and Albright, 2020). As a result, Daxx is not degraded, thereby facilitating histone deposition and assembly of repressive heterochromatin on viral genomes in conjunction with ATRX and the KAP1/SetDB1/HDAC co-repressor complex. Heterochromatin assembly results in repression of the MIEP allowing for the establishment of latency (Murphy et al., 2002; Groves et al., 2009; Sourvinos et al., 2014; Lee et al., 2015; Rauwel et al., 2015; Albright and Kalejta, 2016; Kalejta and Albright, 2020). Importantly, pp71 mutants which cannot interact with Daxx are also unable to activate an MIEP reporter (Hofmann et al., 2002), substantiating the importance of pp71-Daxx interaction for Daxx repression and transactivation from MIEP. Apart from Daxx degradation, pp71 also triggers Daxx SUMOylation (Hwang and Kalejta, 2009). SUMOylated Daxx is unable to interact with NFκB and therefore fails to prevent NFκB acetylation and activation (Croxton et al., 2006; Park et al., 2007; Kim et al., 2017). This leads to NFκB-dependent enhanced MIEP activity (DeMeritt et al., 2004; Liu et al., 2010; Yuan et al., 2015) and efficient IE gene expression at least in certain contexts.

Although the pro-viral relevance of pp71 in initiating viral IE gene expression to induce a de novo lytic infection is well studied, importance of pp71 in mediating viral rejuvenation from latency and its role of NB remodeling in this process remains questionable (Kalejta and Albright, 2020). Interestingly, a recent report has highlighted disruption of PML NBs in a pp71-independent manner by the viral LUNA protein (Poole et al., 2018). LUNA is encoded from a transcript antisense to the viral UL81 and UL82 genes (which also codes for pp71) (Bego et al., 2005) and is expressed not exclusively during latency but also during lytic infection (Keyes et al., 2012). LUNA has been shown to promote HCMV reactivation (Keyes et al., 2012) by triggering dismantling of PML NBs through a proposed deSUMOylase activity (Figures 3, 4; Poole et al., 2018). Mechanistically, LUNA was proposed to act as a cysteine protease, thereby catalyzing SUMO deconjugation from PML and possibly explaining the loss of PML NB integrity in presence of this HCMV protein (Figure 3). Substituting a potential cysteine residue in the predicted catalytic center completely abrogated isopeptidase activity as well as PML NB disruption by LUNA and this phenomenon coincides with impaired viral reactivation from latency. Of interest, reactivation deficiency of LUNA-null or LUNA catalytically dead virus mutants is rescued in PML-deficient cells, further substantiating LUNA-dependent deSUMOylase activity on PML to be important for HCMV rejuvenation from latency (Poole et al., 2018).

During a productive lytic infection, tegument-delivered pp71 initiates and sustains MIEP transcription long enough for the de novo synthesized IE1 protein to promote prolonged lytic phase gene expression (Vardi et al., 2018) and the completion of productive replication. IE1 protein employs a SUMO-dependent mechanism to disrupt PML NBs (Figure 3). IE1 accumulates at PML NB foci and depletes specifically the SUMO-modified isoforms of PML and Sp100 in a proteasome-independent manner without perturbing global SUMOylation (Figure 3; Müller and Dejean, 1999; Xu et al., 2001; Lee et al., 2004; Tavalai et al., 2011; Everett et al., 2013a; Scherer et al., 2013). IE1 itself is SUMOylated on K450 (Xu et al., 2001; Lee et al., 2004; Nevels et al., 2004; Sadanari et al., 2005), but the functional significance of this modification is poorly understood. Expression of an IE1 K450R mutant shows decreased transcripts and protein expression of IE2, the primary viral transactivating protein for HCMV early and late gene expression, suggesting SUMO modification of IE1 to possibly impact IE2 expression (Nevels et al., 2004). Interestingly, SUMOylation of IE1 is dispensable for its PML NB disrupting function (Xu et al., 2001; Spengler et al., 2002; Lee et al., 2004). However, this disruption requires a direct physical interaction between an extended interaction interface of IE1 globular core domain and the coiled-coil domain of PML (Ahn et al., 1998; Lee et al., 2004; Scherer et al., 2014). How IE1 triggers loss SUMO-conjugated isoforms of PML and Sp100 has recently been elucidated in both cell-based assays and in vitro SUMOylation assay. IE1 was found to sensitize only the de novo SUMOylation at K160 of PML after binding to PML but not the deSUMOylation of pre-SUMO-modified PML (Schilling et al., 2017). Moreover, IE1 also prevented As₂O₃-mediated hyperSUMOylation of PML at K160, thereby blocking PML degradation. Since IE1 did not interfere with the coiled-coil-mediated PML dimerization, it has been speculated that IE1 impairs PML autoSUMOylation either by directly abrogating the E3 ligase function of PML (Figure 4) or by preventing access to SUMO sites (Schilling et al., 2017). Besides PML and Sp100, IE1 also targets Daxx to antagonize Daxx-mediated transcriptional repression of the viral genome (Reeves et al., 2010). However, the mechanistic details of IE1-Daxx interaction has not been addressed so far. Moreover, IE1 also induces degradation of SUMO unmodified Sp100 in a proteasome-sensitive way in late stages of infection. This event was revealed to be independent of PML NB disruption (Kim et al., 2011).

Interestingly, IE1-mediated PML NB disruption not only has antiviral significance from the perspective of disarming host cellular intrinsic defense measures but also has implications in abrogating innate immune response (Kim and Ahn, 2015; Scherer et al., 2015). SUMOylation status of IE1 regulates IE1-dependent inhibition of innate immune signaling. Unmodified IE1 interacts with STAT1 and STAT2 in the nucleus and decreases binding of these transcription factors to target promoters, leading to repression of ISG transcription (Paulus et al., 2006; Huh et al., 2008; Krauss et al., 2009). Therefore, in comparison to wild-type HCMV, IE1-deleted HCMV shows hypersensitivity to IFN. Interestingly, SUMOylated IE1 has reduced affinity for STAT2 and is less efficient in inhibiting STAT2-dependent ISG transcription (Huh et al., 2008). In addition to STATs, IE1 also antagonizes type I IFN signaling by interacting with PML, thereby sequestering ISGF3 into a functionally inactive complex incapable of DNA binding and ISG transactivation (Kim and Ahn, 2015).

Structural characterization of the IE1 core (Scherer et al., 2014) has revealed it to be a compact, contiguous domain that is highly sensitive to small deletions and point mutations. A single leucine-to-proline exchange (L174P) within the core domain of IE1 at residue 174 hampers the structural integrity and stability of the protein. Consequently, the L174P mutant is incapable of triggering PML NB disruption and has a growth defect that phenocopies IE1-deleted virus (Scherer et al., 2015, 2017). Only very recently, a novel IE1 mutant (IE1cc172–176) has been characterized, which is selectively deficient in interacting with PML, inhibiting PML SUMOylation, and therefore incapable of PML NB localization and disruption without losing overall structural stability (Paulus et al., 2020). Very interestingly, this mutant has been shown to support viral replication almost as efficiently as its wild-type counterpart. Subsequent functional analysis of IE1cc172–176 revealed that this protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. IE1 mutant which cannot bind to nucleosome (IE1dl476–491) and show reduced chromatin association was revealed to be poorly SUMO-modified, possibly suggesting that IE1 SUMOylation takes place on chromatin. The chromatin-associated E3 SUMO ligase PIAS1 was proposed to SUMOylate IE1 (Paulus et al., 2020). Indeed, PIAS1 has been reported to interact with IE1, to enhance IE1 SUMOylation (Kim et al., 2014), and to act as a SUMO E3 ligase for many transcription factors and chromatin associated proteins (Wu et al., 2014). It was speculated that PIAS1-directed SUMOylation might promote dissociation of the viral protein IE1 from chromatin surface (Paulus et al., 2020). This also explains why IE1 is SUMOylated at nucleosomes but localizes throughout the nucleus. Of interest, with the use of this selective IE1 mutant, not only HCMV IE1-induced deSUMOylation of Sp100 was uncoupled from that of PML, but also upregulation of IFN signaling and ISG expression were observed (Paulus et al., 2020). However, speculation such as disruption of PML NBs upon viral infection is linked to activation rather than inhibition of innate immunity is subject to further studies in other virus infection scenario.

Promyelocytic leukemia and PML NBs have also proven to be critical regulators of the betaherpesvirus human herpesvirus 6A and B (HHV-6A/B). HHV-6 infects peripheral blood mononuclear cells (PBMCs) and various T-cell lines where it can either establish latent infection or can replicate as a part of its lytic programme. Notably, HHV6 integrates its genome into the telomeric regions of host chromosomes for prolonged maintenance. Importantly, PML NBs and the SUMO machinery were shown to be involved in controlling viral latency as well as viral integration. In case of HHV6A, it was demonstrated that knocking down components of PML NB such as PML, Daxx, and Sp100 significantly increased viral lytic replication and late gene expression. Consistently, progressive lytic infection was found to reduce the number of PML NBs within infected cells with concomitant increase in the puncta size without triggering complete disassembly of these speckled structures (Sanyal et al., 2018). These findings are in line with a role of PML NBs in restricting lytic replication of HHV-6A.

Work on HHV-6B has recently revealed a surprising facet of PML and the SUMO machinery in integrating the HHV-6B genome into the telomeric regions of host chromosomes for prolonged maintenance (Collin et al., 2020). Further, the HHV-6B trans-activator protein IE1 which undergoes SUMOylation at K802 and localizes to PML NBs has emerged as the viral regulator in this process (Figure 3; Gravel et al., 2002, 2004; Stanton et al., 2002; Collin et al., 2020). Mechanistically, IE1 co-localizes with all the nuclear PML isoforms to subsequently get hyperSUMOylated by SUMO1 (involving K802 and possibly other SUMO conjugation sites), but not SUMO2/3, in presence of PML and intact PML NBs. A putative SIM as well as the primary SUMO accepting site K802 proved to be essential for IE1 hyperSUMOylation and PML NB recruitment. Moreover, PML was also found to mediate IE1 localization to telomeres and PML depletion sensitized telomeric recruitment of IE1 as well as HHV-6B genomic integration into the host chromosomes (Collin et al., 2020).

Kaposi’s sarcoma-associated herpesvirus is a gammaherpesvirus which is capable of establishing latent infection and can get periodically activated to undergo lytic replication and production of progeny viruses. Much alike to the other herpesviruses, PML NB-mediated restriction of the infecting viral genome has been shown to be important for triggering quiescence of KSHV genome. Latency-associated nuclear antigen LANA1 is the primary viral product during latency and has been reported to bind to a series of cellular gene promoters to modulate gene transcription. LANA1 promotes SUMOylation of Sp100 leading to its aggregation into PML NBs (Figure 3; Günther et al., 2014), thereby fostering structural integrity of these structures. LANA1 has a SUMO2-specific SIM which aids in SUMOylation of LANA1 at K1140 residue and also enables recruitment of SUMO2-modified KAP1 together with the Sin3A-containing transcriptional repressor complex. LANA1-KAP1 association maintains viral latency by silencing the expression of viral lytic trans-activator K-Rta. Deletion of SIM from LANA1 abrogates this association, thereby triggering loss of viral episomal genome maintenance and loss of repression of K-Rta expression (Cai et al., 2013). A recent study also demonstrated that inhibition of the LANA SIM motif by a small molecule Cambogin exerted significant antagonistic effects on KSHV persistent infection and proliferation of KSHV-latently infected cells both in vitro and in vivo (Ding et al., 2019). Notably, LANA1 also exerts E3 SUMO ligase-like activity (Figure 4) by enhancing SUMOylation of histones H2A-H2B in a SIM-dependent manner through recruiting the SUMO-Ubc9 intermediate. This likely contributes to chromatin condensation, repression of viral genes and maintenance of viral latency (Campbell and Izumiya, 2012). LANA1 SUMOylation is controlled by the SUMO isopeptidase SENP6 and LANA1 and SENP6 expression are mutually interdependent (Lin et al., 2017). SUMOylated LANA1 further increases LANA1 expression whereas SENP6-mediated deSUMOylation reduces LANA1 expression. Interestingly, LANA1 itself binds to SENP6 promoter to reduce SENP6 expression. This leads to an increased pool of SUMO-modified LANA1 which further maintains its high expression and promotes viral latency. During de novo infection, overexpressing SENP6 has been shown to impair viral latency owing to diminished LANA protein levels which further led to enhanced viral gene expression (Lin et al., 2017).

Another KSHV protein K-bZIP that belongs to the basic region-leucine zipper family of transcription factors, usurps host cellular SUMOylation machinery to exert its function (Izumiya et al., 2005). K-bZIP acts as a transcriptional repressor of the viral genome and aids in maintaining viral latency. Interestingly, K-bZIP has been shown to interact with Ubc9 and to potentially serve as a viral SUMO E3 ligase (Figure 4). K-bZIP’s proposed SUMO E3 ligase activity was revealed to be specific for SUMO2/3 conjugation, possibly implicating a role in promoting polySUMOylation of targets. K-bZIP is itself modified at K158 both in vitro and in vivo (Chang et al., 2010); but a SUMO-deficient mutant of K-bZIP does not show altered protein stability or localization to PML NBs. Further, SUMOylation of K-bZIP is mostly dispensable for the trans-repression activity. However, trans-repression requires interaction of K-bZIP with the catalytically active Ubc9 (Izumiya et al., 2005). Though detailed molecular explanations are lacking, K-bZIP has been shown to induce SUMO2/3 conjugation of transcription factors at the viral promoters, resulting in repression of viral transcription and establishment of viral latency (Yang et al., 2015a). During KSHV reactivation, enrichment of SUMO2/3 association with the promoter regions of both the viral and cellular genomes has been reported (Chang et al., 2013; Yang et al., 2015a). Though awaiting experimental affirmation, K-bZIP may act as a SUMO conjugating agent at cellular promoters thereby repressing cellular innate immune response (such as expression of ISGs) (Chang et al., 2013). At viral genomes, K-bZIP fosters enhanced association of SUMO2/3 with the viral DNA, thereby providing another check for the virus to skew its life cycle back toward latency before lytic commitment. Infection with a virus harboring a SUMO E3 ligase mutant of K-bZIP does not show this effect and has increased proliferation capabilities. Interestingly, knocking down SUMO2/3 expression phenocopied this effect (Yang et al., 2015a). Of note, K-bZIP also harbors a SIM that binds exclusively to SUMO2/3 (Chang et al., 2010). Direct implications of K-bZIP in usurping PML NB components for SUMOylation to induce KSHV latency, however, are lacking so far.

Kaposi’s sarcoma-associated herpesvirus employs several strategies to target PML NB integrity for inducing viral reactivation and lytic rejuvenation. The first KSHV protein which can antagonize PML NBs during de novo infection is one of the viral tegument proteins ORF75 which specifically targets ATRX for degradation in a proteasome-independent manner and triggers redistribution of Daxx, without hampering other PML NB constituents (Figure 3; Full et al., 2014). PML NBs become lesser in number and appear as irregular-shaped larger structures in cells where KSHV-ORF75 is expressed alone. A mutant KSHV lacking the entire ORF75 gene is replication-deficient in cell culture and cannot promote viral gene expression, though much of it can be attributable to the importance of this KSHV protein in structural integrity of the virus (Full et al., 2014).

A robust SUMO-dependent way that KSHV adopts to disrupt PML NB aggregates during the onset of lytic replication requires the viral protein K-Rta. This KSHV trans-activating protein exerts StUbL activity by targetting SUMOylated proteins for ubiquitylation and proteasomal degradation (Figures 3, 4; Izumiya et al., 2013). K-Rta possesses a RING finger domain and multiple SIMs with affinity toward SUMO2/3 multi-/polymers (Figure 4). SUMOylated PML is one of the primary targets of K-Rta, explaining the disruption of PML NBs by K-Rta (Figure 3). Accordingly, SIM-mutated K-Rta was shown to be incapable of triggering PML NB disruption (Izumiya et al., 2013). Another important target of K-Rta is K-bZIP. K-bZIP acts as a transcriptional repressor of K-Rta, thereby impairing viral transactivation and lytic rejuvenation. Therefore, degradation of K-bZIP by the StUbL activity of K-Rta counteracts the repressive action of K-bZIP. Indeed, SIM-mutated K-Rta showed reduced replication potential and attenuated potency of viral transactivation by K-Rta (Izumiya et al., 2013).

Another KSHV protein which disrupts PML NBs is LANA2 (vIRF-3). LANA2 promotes PML SUMOylation and induces proteolytic ubiquitylation followed by proteasomal demise of SUMO-enriched PML, again leading to dissolution of PML NBs (Figure 3; Marcos-Villar et al., 2009, 2011). Mutation of either the SUMO conjugation site of PML or deletion of SIM within LANA2 significantly impaired PML NB disruption, but the mechanism of this process has remained unclear (Marcos-Villar et al., 2009, 2011). Since LANA2 lacks typical features of a ubiquitin ligase, intrinsic StUbL activity of LANA-2 is unlikely. In addition to the significance of SIM, SUMOylation of LANA2 at multiple K residues are also found to be required for this PML NB disruptive function (Marcos-Villar et al., 2009, 2011). Notably, as LANA2 is a viral latency associated protein, the functional relevance of PML NB disruption by a viral latent protein has not been ascertained experimentally. Altogether, KSHV adopts multiple SUMO-dependent strategies to usurp PML NBs for maintaining latency and further disrupts their structural integrity for rejuvenation and lytic proliferation.

Epstein-Barr virus, a gammaherpesvirus with a double stranded DNA genome, can cause B cell lymphoma, nasopharyngeal cancer, Hodgkin’s and non-Hodgkin’s lymphomas, and a subset of gastric cancers. It can either be maintained in a latent state and lead to unrestricted cell proliferation and tumorigenesis or can switch to lytic proliferation, especially in the infected epithelial cells. Interestingly, the SUMO system may be involved in maintenance of EBV latent infection. The primary EBV oncoprotein latent membrane protein 1 (LMP1), which is pivotal in modulating viral quiescence, interacts with Ubc9 leading to SUMOylation of many host proteins (Li and Chang, 2003; Bentz et al., 2011). Two important targets of LMP1-mediated SUMOylation are IRF7 and KAP1 (Bentz et al., 2012, 2015). SUMO-conjugated IRF7 at K452 has increased nuclear localization and stability but decreased transactivation potency and therefore is incapable of mounting a robust innate immune response in cells latently infected with EBV (Bentz et al., 2012). SUMOylation of KAP1 increases its transcriptional repression on EBV early promoters and the lytic origin of replication of the virus (Bentz et al., 2015), thereby decreasing transcription of Zta and Rta (EBV lytic trans-activators) and contributing to viral latency (Bentz et al., 2015). None of the LMP1-directed SUMOylation targets resides within PML NB, but LMP1 increases PML expression and immunofluorescence intensity of PML NBs. Disruption of PML NBs results in EBV lytic replication, suggesting that NB disruption is involved in the switch from latent to lytic infection (Sides et al., 2011a, b).

The EBV tegument protein BNRF1 can target PML NBs by interacting with Daxx and disrupting the formation of the Daxx-ATRX Histone H3.3 chaperone complex (Figure 3). Mechanistically, BNRF1 binds to Daxx and outcompetes binding of Daxx to ATRX. Subsequent failure of Histone H3.3 loading onto the viral chromatin leads to enhancement of viral gene expression during primary infection (Tsai et al., 2011, 2014). In addition to preventing Daxx/ATRX/Histone H3.3-mediated heterochromatinization of the viral genome, BNRF1 also facilitates formation of the stabilized ternary complex with Daxx, and histone variants H3.3/H4 and alters the histone dynamics in favor of expression of viral genes at pre-latency stage (Tsai et al., 2014). This further allows expression of latency associated genes and establishment of latent infection.

Disruption of the integrity of PML NBs is crucial for lytic rejuvenation of EBV (Bell et al., 2000). The EBV protein EBNA1 induces degradation of PML through the ubiquitin-proteasome system (Figure 3; Sivachandran et al., 2008). Degradation involves binding to the deubiquitylase USP7 and the kinase casein kinase 2 (CK2), which promotes phosphorylation and subsequent degradation of PML. Disruption of PML NBs also impairs p53 activation, thereby likely contributing to tumorigenesis (Sivachandran et al., 2008, 2010). Interestingly, PML disruption by EBNA1 occurs in epithelial cells, which promote lytic infection, but has not been observed in B lymphocytes, which are predominantly responsible for latent infection, further supporting the idea that dismantling of NBs predisposes latently infected cells to lytic rejuvenation (Sivachandran et al., 2012). Consistently, depleting PML was shown to trigger EBV lytic cycle and supplementing PML depleted cells with single PML isoform (PML I-VI) suppressed EBV lytic reactivation except in case of PML IV which was revealed to be the most vulnerable PML isoform to EBNA1-mediated degradation (Sivachandran et al., 2012).

An immediate-early protein of EBV Zta (BZLF1), which is involved in lytic replication of the virus and transactivation of EBV early genes, can also disrupt PML NBs even under exogenously expressed condition (Figure 3; Adamson and Kenney, 2001). Zta itself can be SUMOylated at K12 (Adamson and Kenney, 2001; Hagemeier et al., 2010; Murata et al., 2010) and it has been proposed that Zta inhibits SUMO conjugation to PML by competing for the limiting pool of endogenous SUMO. Loss of SUMO-modified PML induces the dissolution of PML NBs and a diffuse nuclear distribution of PML (Figure 3; Adamson and Kenney, 2001). Notably, however, partial disruption of PML NBs was still observed upon expression of a SUMOylation-deficient mutant of Zta, implying other viral regulators to have PML NB disruptive functions (Hagemeier et al., 2010). Consistently, another study revealed overexpression of many EBV-encoded proteins such as BDLF1, EBNA3B, BRLF1, BFLF2, BLLF2, and BZLF1 to have the potency to reduce the average number of PML NBs in infected cells (Salsman et al., 2008). Therefore, EBV has developed multiple mechanisms to disintegrate PML NB integrity which emphasizes the significance of these structures during the lytic cycle of this gammaherpesvirus.

There are also reports of viral protein such as the EBV-encoded protein kinase (EBV-PK), and host regulatory protein such as the cellular scaffolding protein RanBPM to dictate Zta SUMOylation status and therefore to influence Zta-mediated lytic reactivation of EBV (Hagemeier et al., 2010; Yang et al., 2015b). Whether these regulatory aspects of Zta SUMOylation have any direct impact on Zta’s ability to disrupt PML NBs has not been addressed yet.

Therefore, in general, herpesviruses exploit the molecular assembly within PML NBs to heterochromatinize their genome and to go into the latent phase of infection. During rejuvenation, they actively disrupt PML NB integrity by specific lytic proteins to trigger lytic proliferation.

In contrast to the early recruitment of herpesviral DNA to PML NBs, adenoviral DNA is associated with PML NBs at a relatively late stage of infection (≥4 h) (Komatsu et al., 2015). Interestingly, this targeting does not require the incoming viral DNA itself but is mediated by the viral single-strand DNA binding protein DBP (also called E2A) which is involved in viral DNA replication (Komatsu et al., 2015). During infection with adenovirus type 5, constituents of PML NBs, including PML and Daxx, are re-organized from their typical punctate appearance to elongated nuclear tracks (Figure 3; Carvalho et al., 1995; Puvion-Dutilleul et al., 1995). Mechanistically, this reorganization involves an interaction of the adenoviral protein E4orf3 with the PML (Figure 3; Doucas et al., 1996; Hoppe et al., 2006). Elegant structural work revealed that E4orf3 forms a multivalent polymer thereby creating avidity-driven interactions with PML and other components of these tracks (Ou et al., 2012). Reorganization of PML NBs fosters viral replication as an E4orf3 mutant that is defective in reorganization is severely growth-compromised. Further, this E4orf3 mutant virus is less potent in antagonizing the IFN-induced antiviral state. Interestingly, however, this phenotype can be rescued upon depletion of PML, underlining the importance of PML in anti-viral defense (Ullman et al., 2007; Ullman and Hearing, 2008). E4orf3-induced nuclear tracks also sequester other antiviral host proteins, such as Mre11 and Nbs1, which are part of the MRN (Mre11-Rad50-Nbs1) complex that mediates the DNA damage response (DDR) and DNA repair (Sohn and Hearing, 2012). The DDR hampers viral DNA replication and HAdV has evolved complementary mechanisms to inhibit the MRN complex. In addition to the sequestration of MRN components within the tracks of infected cell nuclei, the virus mediates ubiquitin-proteasome-dependent degradation of Mre11 through an E3 cullin-RING ligase (CRL) complex assembled by adenoviral proteins E1B-55K in conjunction with E4orf6. Importantly, E4orf3-mediated track sequestration of Mre11 and Nbs1 triggers their SUMOylation potentially facilitating their relocation to cytosolic aggresomes and subsequent degradation (Stracker et al., 2002; Araujo et al., 2005; Liu et al., 2005; Karen et al., 2009; Sohn and Hearing, 2012). Along this line, the transcriptional regulators TIF1γ and TFII-I are also sequestered in PML containing nuclear tracks, where they undergo E4orf3-mediated polySUMOylation and subsequent ubiquitin-proteasom-dependent degradation, most likely through cellular StUbL activity (Bridges et al., 2016; Sohn and Hearing, 2016). Removal of TIF1γ and TFII-I might cause de-repression of specific adenoviral promoters, thereby favoring viral gene transcription. The inactivation of antiviral host factors by virus-induced sequestration or proteasomal degradation is also exemplified by inhibition of Daxx function (Figure 3; Schreiner et al., 2010). In this case it has been proposed that, the adenoviral capsid protein VI interacts with and displaces Daxx from PML NBs to the cytoplasm, thereby de-repressing Daxx-mediated silencing of the viral E1A promoter (Figure 3; Schreiner et al., 2012). Further, SUMO conjugated E1B-55K in conjunction with the cellular StUbL RNF4 was shown to induce proteasomal degradation of Daxx (Endter et al., 2001; Schreiner et al., 2011; Wimmer et al., 2013; Müncheberg et al., 2018). Altogether, these data indicate that reorganization of PML NBs and E4Orf3-mediated formation of tracks in the course of adenovirus infection is a key process in repurposing or inactivating cellular regulators of transcription and DNA repair. Notably, these nuclear tracks also harbor PIAS3 SUMO E3 ligase which might further explain the stimulatory effect of E4orf3 on SUMO conjugation and SUMO chain formation on many host proteins (Higginbotham and O’Shea, 2015; Sohn et al., 2015). Alternatively, E4ORF3 functions as a SUMO E3 ligase and/or E4 chain elongase on selected substrates (Sohn and Hearing, 2016).

Interestingly, the E4orf3-positive tracks reside in close spatial proximity of the adenoviral replication centers (RCs) and a recent report has unveiled the pivotal role of SUMOylation for this positioning (Stubbe et al., 2020). In particular, E2A was found to undergo SUMO conjugation, thereby fostering interaction with both PML and Sp100A and convergence of PML NB tracks with the adenoviral RCs (Figure 3). A SUMOylation-deficient E2A mutant exhibits reduced binding to PML and Sp100A, and accordingly, viral RCs and PML tracks remain separated. Compared to their wild type counterpart these mutant viruses are less efficient in yielding progeny virions (Stubbe et al., 2020). Juxtaposition of viral RCs to PML NBs may promote adenoviral early gene transcription through PML-mediated recruitment of the viral E1A-13S protein (Figure 3; Berscheminski et al., 2013). The compartmentalization of PML NB components within the adenoviral RCs or E4orf3-containing tracks is a very selective process that is, at least partially, regulated by SUMO-dependent mechanisms (Berscheminski et al., 2014). Sp100B, Sp100C, and Sp100HMG are delocalized to the adenoviral RCs possibly as part of a viral strategy to counteract the restrictive functions of these specific Sp100 isoforms on chromatin architecture. Sp100A, on the other hand, is retained around the periphery of the RCs as a part of the non-canonical track-like PML NB structures, where it favors transcriptional activation of the adenoviral genome (Berscheminski et al., 2014). Mechanistically, adenoviral infection triggers loss of SUMO2 conjugated Sp100A leading to reduced association between Sp100A and HP1, explaining chromatin decondensation by Sp100A on adenoviral promoters (Berscheminski et al., 2014).

Apart from E4orf3, E1B-55K also exploits the SUMO system to target host cell proteins. E1B-55K itself is modified by SUMO and the modification facilitates its nuclear retention by blocking a CRM-dependent nuclear export signal (Kindsmüller et al., 2007; Wimmer et al., 2013). Further, SUMOylation of E1B-55K is important for its interaction with PML IV (Figure 3; Wimmer et al., 2010). Interestingly, E1B-55K can also induce SUMOylation of several PML NB-associated host proteins, including p53 and Sp100. Inhibition of p53 activity by E1B-55K is a hallmark of HAdV infection and E1B-55K appears to apply different strategies to inhibit the antiproliferative and pro-apoptotic activity of p53. One strategy is the ubiquitin-proteasome-mediated degradation of p53 by the above-mentioned CRL/E1B-55K/E4orf6 ubiquitin ligase complex (Figure 3; Querido et al., 2001). Intriguingly, E1B-55K also induces SUMOylation of p53 thereby rendering it transcriptionally inactive by facilitating its nuclear export through transient interactions with PML NBs (Figures 3, 4; Muller and Dobner, 2008; Pennella et al., 2010). Whether E1B-55K exerts E3 SUMO ligase activity by its own or stimulates SUMOylation by co-operating with host E3 ligases is a matter of debate (Muller and Dobner, 2008; Pennella et al., 2010). In addition to p53, E1B-55K also triggers SUMOylation of Sp100A and sequesters it within PML containing nuclear tracks (Figures 3, 4), thereby limiting stimulatory effects of Sp100A on transactivation potential of p53 (Berscheminski et al., 2016). Moreover, ATRX is targeted by the CRL/E1B-55K/E4orf6 complex for proteasomal degradation (Figure 3; Schreiner et al., 2013).

The adenoviral protein E1A also has PML NB destabilizing properties (Figure 3). The mechanism involves a direct interaction between the conserved region 2 of E1A and the N-terminal region of Ubc9 which binds SUMO and regulates polySUMOylation (Figures 3, 4; Yousef et al., 2010). Therefore, interaction of Ubc9 with E1A might affect polySUMOylation, which might further destabilize PML NB integrity (Figure 3; Yousef et al., 2010). At least in a polySUMOylation assay in yeast, wild-type E1A, but not a mutant defective in Ubc9 binding, inhibited polySUMOylation. Moreover, the authors also speculated that E1A binding might affect the ability of Ubc9 to be SUMOylated at K14 residue. As SUMO modification at this residue affects Ubc9 substrate specificity, this interaction might also alter SUMOylation of host proteins in general (Yousef et al., 2010).

Therefore, HAdV selectively usurps certain components of PML NB components at the expense of antagonizing others to favor its own perpetuation.

Chicken embryo lethal orphan adenovirus adopts a robust SUMO-dependent mechanism of PML NB disruption. The viral early protein Gam1 induces ubiquitin-proteasome-dependent degradation of the dimeric SUMO activating enzyme SAE1/SAE2 (Figure 4) by recruiting a cullin-RING E3 ubiquitin ligase (Cul2/5- Rbx1-Elongin B/C) complex (Boggio et al., 2004, 2007). The resulting loss of SUMO conjugation is transduced to the disruption of PML NBs possibly as a result of absence of PML SUMOylation (Colombo et al., 2002; Boggio et al., 2007). As SUMOylation usually leads to transcriptional repression, this global loss of SUMOylation might have a pro-viral significance (Boggio et al., 2004).

Though not as elaborately studied as DNA viruses, there are examples of RNA viruses interacting with the host cellular SUMOylation machinery and PML NBs. One classical example includes anti-PML activities of encephalomyocarditis virus (EMCV) protease 3C. EMCV which belongs to the family Picornaviridae can cause neurological and reproductive complicacies in many mammals, likely including humans through zoonosis. Interestingly, primary fibroblasts derived from PML knockout mice are more vulnerable to infection with EMCV, suggesting that the PML protein might act as a restriction factor for EMCV replication (El McHichi et al., 2010). Moreover, progressive infection with EMCV was found to cause a reduction in PML protein levels both in IFN-treated cells and in PML III-expressing cells. The degradation event was preceded by translocation of PML from nucleoplasm to the nuclear matrix and SUMO1, SUMO2/3 conjugation to PML, leading to an increase in PML NB size. Subsequently, the viral protease 3C was found to localize to the PML NBs along with proteasome components to trigger PML degradation in a proteasome- and SUMO-dependent manner (Figure 3); PML SIM, however, proved to be dispensable for the degradation (El McHichi et al., 2010). Unlike PML III which is antagonized by EMCV protease 3C, SUMO-conjugated PML isoform IV has been reported to interact with the EMCV 3D polymerase via the isoform’s unique C-terminal domain and to sequester the viral protein to PML NBs (Figure 3), thereby impairing viral perpetuation (Maroui et al., 2011). Consistently, selective depletion of PML IV also undermines the anti-EMCV potency of IFN (Maroui et al., 2011).

SUMOylation of PML has been reported to have indirect effects on the life cycle of poliovirus, another Picornaviridae family member etiologically responsible for paralytic poliomyelitis. The mechanism includes virus-mediated activation of the extracellular-signal-regulated kinase (ERK), which phosphorylates PML, thereby facilitating PML SUMOylation and translocation to the nuclear matrix. Subsequently, p53 gets recruited to PML NBs and becomes phosphorylated on Ser15 in a PML-dependent way (Figure 3; Pampin et al., 2006). Therefore, poliovirus infection results in PML-dependent p53 activation and mobilization of apoptotic cascade downstream of activated p53, resulting in inhibition of viral replication. PML III overexpression conferred resistance to poliovirus in p53 wild-type cells, but not in p53-inactive cells, while depletion of PML III abolishes p53-dependent apoptosis and antiviral effects. Interestingly, as a countermeasure against this transient cyto-protective effects, poliovirus induces degradation of p53 through proteasome via the E3 ubiquitin ligase Mdm2 (Pampin et al., 2006).

Antiviral effects of PML have also been extended to VSV, a member of the Rhabdoviridae family which infects human zoonotically, as exogenous PML III conferred resistance to VSV by inhibiting viral mRNA and protein synthesis (Chelbi-Alix et al., 1998), while PML knockout mice were found to be more susceptible to VSV infection than wild type mice (Bonilla et al., 2002). Disruption of PML NBs, however, has not been observed in VSV infected cells. The mechanism of how PML exerts antagonistic activity against a virus, whose replication takes place entirely in the cytoplasm and which does not disrupt PML NBs, is yet to be addressed. The observation that only the coiled-coil domain deletion mutant of PML, but not the other RING finger PML mutant and cytoplasmic PML III mutant, fails to show the antiviral activity, emphasizes the importance of the coiled-coil region of PML for anti-VSV effects (Chelbi-Alix et al., 1998). Another report has also elucidated PML IV to have anti-VSV activity (El Asmi et al., 2014). Mechanistic profiling elucidated PML IV to potentiate both the intrinsic and innate immune arms of the antiviral defense pathway. The innate immune response involves SUMOylated PML IV to interact with and recruit peptidyl-prolyl cis/trans isomerase (Pin1) to PML NBs. This results in activated IRF3 to escape from Pin1-mediated degradation and to potentiate its canonical functions of IFN-β synthesis. Interestingly, though depletion of IFR3 abrogated PML IV-induced IFN synthesis, it could not entirely inhibit PML IV-induced inhibition of viral proteins, suggesting parallel IFN-dependent and independent pathways to operate downstream of PML IV. Moreover, the agonistic effects of SUMOylated PML IV to stimulate IFN-β synthesis was found to be a broader effect on innate immune regulation rather than a VSV infection-specific scenario (El Asmi et al., 2014).

Unlike VSV, another Rhabdoviridae family member rabies virus which causes central nervous system dysfunctions does reorganize PML NBs during infection to form larger and more electron-dense aggregates. The P3 protein, an amino-terminally truncated product of the viral P mRNA which also codes for phosphoprotein P, causes this effect. Interestingly, sole expression of P delocalizes PML III from nuclear to cytoplasmic puncta where both proteins colocalize (Figure 3). A direct interaction was also evidenced between the C-terminal domain of P protein and the PML RING finger. However, the observations that overexpression of PML III isoform does not perturb rabies infection but that PML^–/^– MEFs are more susceptible to rabies infection than wild-type MEFs, do suggest involvement of other PML isoforms to take part in the anti-rabies defense (Blondel et al., 2002). Indeed, subsequent studies revealed the antiviral importance of PML IV, but not of other nuclear as well as cytoplasmic PML isoforms, to act as a restriction factor against rabies infection independent of IFN signaling axis. Notably, PML IV SUMO conjugation was found to be essential for its antiviral effect as the protective effect of PML IV was lost when the SUMOylation sites were mutated (Blondel et al., 2010).

The RING finger protein Z of LCMV, an Arenaviridae family member which transmits zoonotically to human leading to aseptic meningitis, induces redistribution of PML from nuclear condensates to cytoplasmic aggregates during infection (Figure 3). In these aggregates, both proteins interact with the translation initiation factor eukaryotic initiation factor 4E (eIF-4E) and reduce eIF-4E’s affinity for the 5′ mRNA cap structure, thereby inhibiting cellular translation (Borden et al., 1998; Kentsis et al., 2001). Subsequently, PML has been shown to act as a restriction factor during infection with LCMV as PML knockout mice showed more susceptibility to LCMV infection than their wild type counterparts (Bonilla et al., 2002). Consistently, anti-LCMV potency of IFN was also found to be partially sensitive to PML knocking out in MEFs, suggesting PML to be one of the IFN-induced antiviral mediators for defense against LCMV (Djavani et al., 2001). However, neither overexpression of PML III nor Sp100 had any repressive effect on LCMV growth (Asper et al., 2004), implicating PML isoforms other than PML III to have anti-LCMV relevance.

Like other RNA viruses, PML exerts antagonistic effects during infection with the Orthomyxoviridae family member influenza A virus in a viral strain-specific manner (Li et al., 2009). Overexpression of different PML isoforms such as PML III, PML IV, and PML VI attenuates influenza A viral propagation in cell culture (Chelbi-Alix et al., 1998; Iki et al., 2005; Li et al., 2009), while PML silencing has an agonistic effect (Iki et al., 2005; Li et al., 2009). However, neither the mechanism by which PML exerts anti-influenza activity nor the significance of PML NB recruitment of viral proteins (the matrix protein M1 and the nonstructural polypeptides NS1 and NS2) (Iki et al., 2005) has been addressed yet. A more recent study suggested induction of a specific host cellular SUMOylation response in influenza virus infected cells. However, PML SUMOylation was insensitive to this infection. Interestingly, confocal imaging showed PML-stained puncta to get smaller and more in number whereas Sp100 and Daxx appeared dispersed in the nuclear compartment of infected cells, suggestive of PML NB dispersion (Domingues et al., 2015).

Interplay of HCV, a member of the family Flaviviridae, with the host cellular PML NBs involves the HCV core protein to get recruited to PML NBs by interacting with PML IV and to inhibit the co-activator activity of PML IV on p53 (Figure 3). This leads to prevention of p53-dependent apoptotic induction and development of HCV-associated hepatocellular carcinoma (Herzer et al., 2005). However, SUMO-dependent regulation of PML NB structures has not yet been reported for HCV.

Promyelocytic leukemia has recently been identified as an antiviral host cellular determinant capable of restricting all serotypes of DENV, another Flaviviridae family member, as siRNA-mediated depletion of PML resulted in increased virus production (Giovannoni et al., 2015, 2019). Moreover, as a part of viral countermeasures, PML NBs were found to get significantly reduced in number during infection with DENV serotypes. The causative agent was identified to be the non-structural viral protein NS5 which co-localized with PML III and IV at PML NBs and promoted accelerated degradation of PML III and IV (Figure 3; Giovannoni et al., 2019). Consistently, over expression of these specific PML isoforms curtailed DENV replication and progeny yield. Of note, Sp100 and Daxx were proved to be dispensable for DENV progeny production, suggesting specific anti-DENV implications of PML III and IV (Giovannoni et al., 2019). Interestingly, DENV NS5 interacts with Ubc9 and undergoes SUMOylation. This SUMOylation requires a SIM domain within NS5 (Su et al., 2016). Any correlation of SUMO conjugation of NS5 on its PML NB disruptive property, however, is yet to be addressed experimentally.

During infection with HIV-1, a lentivirus belonging to the family Retroviridae, PML NBs rapidly relocalize from nuclear to cytoplasmic aggregates (Figure 3). Further, PML depletion increased reverse transcription capacity of HIV-1. However, involvement of PML is likely indirect through stabilization of Daxx which sensitized the reverse transcription of HIV-1 RNA by spatially relocating to incoming viral capsids in the cytoplasm. Similar relocation of PML NB components and Daxx-dependent antagonistic impact of PML were also observed in other retroviruses such as simian immunodeficiency virus (SIVmac), murine leukemia virus Moloney and equine infectious anemia virus. Interestingly, SUMOylation inhibitor ginkgolic acid (that impairs the formation of the E1-SUMO intermediates) strongly sensitized formation of cytoplasmic PML-containing aggregates in HIV-1 infected cells, suggesting importance of SUMO conjugation for structural integrity of these cytoplasmic aggregates. However, data based on ginkgolic acid treatment need to be interpreted with a note of caution since it is a rather unspecific SUMO inhibitor. Further, no other specific SUMO target specifically directing this delocalization has been assigned (Dutrieux et al., 2015). An interesting recent report also elucidated another SUMO-dependent PML-NB disruptive strategy by the HIV-1 accessory protein Vpu. In brief, Vpu interacts with RanBP2 at nuclear pore to inhibit the SUMO E3 ligase activity of the RanBP2–RanGAP1^∗SUMO1–Ubc9 complex, thereby affecting the PML NB integrity (Figure 3) and SUMOylation of DNA repair proteins BLM, Rad52. As a consequence, Rad52-dependent homologous repair and circularization of non-integrated linear DNA from a superinfecting virus were prevented. Moreover, BLM-dependent nucleolytic attack on those susceptible DNA ends were facilitated (Volcic et al., 2020). On a different note, PML aggregation was observed in syncytia present in the brain or lymph nodes of infected patients or in syncytia elicited by the envelope glycoprotein complex of HIV-1 in vitro (Perfettini et al., 2009). PML was shown to foster phosphorylation of ATM which was co-recruited with PML in nuclear aggregates and mobilized p53-dependent syncytial apoptosis in HIV-infected CD4 cells (Perfettini et al., 2009). However, any mechanistic role of SUMO-mediated regulation has not been addressed in this respect.

Human foamy virus are retroviruses infecting non-human primates but can be transmitted to human through zoonotic infection. Though endogenous PML has been shown not to interfere with HFV replication and latency and also not to get delocalized during infection (Regad et al., 2001; Meiering and Linial, 2003), PMLIII overexpression reportedly attenuates HFV transcription by interacting with the HFV transactivator protein Tas and hindering its binding to DNA (long-terminal repeat and internal promoter) (Regad et al., 2001). However, this interaction was revealed to be independent of SUMO regulation. Anti-viral potency of IFN is also diminished in PML deficient MEFs but other IFN-dependent antiviral effectors such as MxA, Mx1, and PKR could not sensitize HFV replication (Regad et al., 2001), again bolstering the specificity of PML in creating an IFN-induced antiviral state against HFV.