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Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

['Johannes Schweininger', 'Division Of Biotechnology', 'Department Of Biology', 'Friedrich-Alexander-University Erlangen-Nürnberg', 'Erlangen', 'Myriam Scherer', 'Institute Of Virology', 'Ulm University Medical Center', 'Ulm', 'Franziska Rothemund']

Date: 2021-08

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1 CORE ). We found that IE1 CORE domains, also including the previously characterized IE1 CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1 CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

Cytomegaloviruses have evolved in very close association with their hosts resulting in a highly species-specific replication. Cell-intrinsic proteins, known as restriction factors, constitute important barriers for cross-species infection of viruses. All cytomegaloviruses characterized so far express an abundant immediate-early protein, termed IE1, that binds to the cellular restriction factor promyelocytic leukemia protein (PML) and antagonizes its repressive activity on viral gene expression. Here, we present the crystal structures of the PML-binding domains of rat and human cytomegalovirus IE1. Despite low amino-acid sequence identity both proteins share a highly similar and unique fold forming a distinct protein class. Functional characterization revealed a common mechanism of PML antagonization. However, we also detected that the respective IE1 proteins only interact with PML proteins of the natural host species. Interestingly, expression of HCMV IE1 allows rat cytomegalovirus infection in human cells. This indicates that the cellular restriction factor PML forms an important barrier for cross-species infection of cytomegaloviruses that might be overcome by adaptation of IE1 protein function. Our data suggest that the cytomegalovirus IE1 structure represents an evolutionary optimized protein fold targeting PML proteins via coiled-coil interactions.

In this study, we present the experimentally determined crystal structures of human and rat cytomegalovirus (RCMV) IE1 CORE . All crystallized IE1 CORE domains share a highly similar, all-α-helical fold. Since we observed that the mechanism of PML-NB disruption is likewise conserved between primate and rodent IE1 proteins, we conclude that cytomegaloviruses have evolved this distinct protein fold to effectively bind and inactivate an important antiviral defense. Closer investigation of the crystal structures revealed slight differences in the helix arrangement of rat compared to primate cytomegalovirus IE1. This correlates with a comparative functional analysis of human and rat cytomegalovirus IE1 showing that neutralization of PML-NBs occurs only in cells of the natural host species but not during cross-species infection. For RCMV, this block of cross-species infection can be alleviated by expression of human IE1 in human host cells. In summary, our data provide evidence that the IE1 fold has been adapted to maximize the efficiency of PML-NB targeting and strengthen the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV.

In order to overcome the PML-NB-based defense, herpesviruses encode antagonistic effector proteins, which employ different strategies to either inactivate single PML-NB components or to disrupt the integrity of the whole structure. The herpes simplex virus type I immediate-early protein ICP0, for instance, disarms PML-NBs in a rapid and efficient way by inducing a widespread proteasomal degradation of SUMO-modified proteins including PML-NB components [ 17 ]. In contrast, immediate-early protein IE1 of human cytomegalovirus (HCMV), a ubiquitous β-herpesvirus causing serious disease in immunocompromised individuals, uses a more careful strategy, likely due to the prolonged replication cycle of HCMV. IE1 directly interacts with PML and blocks its SUMOylation in a proteasome-independent manner [ 18 , 19 ]. Since SUMO modification of PML is essential for PML-NB integrity, this results in a dispersal and inactivation of PML-NB foci. Structural characterization of IE1 has shown that it comprises a folded core domain (IE1 CORE ), which mediates the interaction with PML and is flanked by a short disordered region at the N-terminus and a longer disordered region at the C-terminus containing a SUMOylation motif and a STAT interaction site [ 20 – 22 ]. Crystallization of the IE1 CORE domain of rhesus cytomegalovirus (RhCMV), as described in a previous publication of our groups, revealed a so-far unobserved femur-like all-α-helical fold with local similarity to the conserved coiled-coil domain of TRIM proteins [ 22 ]. Since IE1 CORE efficiently binds to the PML (TRIM19) coiled-coil domain, we proposed that IE1 sequesters PML via structural mimicry using an extended binding surface.

Specific structures within the cell nucleus termed PML nuclear bodies (PML-NBs) or nuclear domain 10 (ND10) have been shown to play a major role in the intrinsic defense against a variety of viruses, including members of the highly host-adapted herpesvirus family [ 4 ]. PML-NBs are dynamic multiprotein complexes that accumulate in distinct foci within the interchromosomal space and have been implicated in cellular key processes such as cell cycle progression, apoptosis, senescence, DNA damage and antiviral responses [ 5 ]. PML, the signature protein of PML-NBs, belongs to the immunomodulatory tripartite motif (TRIM) protein family, whose members share an N-terminal domain structure comprising a RING domain, one or two B-Boxes, and a coiled-coil (CC) domain (often subsumed under the term RBCC domain) [ 6 ]. Within the N-terminal region, PML additionally harbors target sites for covalent modification with small ubiquitin-like modifier (SUMO) proteins, which enables the interaction with further protein components and, therefore, is essential for PML-NB biogenesis [ 7 , 8 ]. Upon herpesvirus infection, PML-NBs associate with viral genomes as soon as they have entered the nucleus [ 9 , 10 ]. This association blocks viral infection at a very early step, since PML-NB proteins rapidly promote the condensation of herpesviral DNA into transcriptionally inactive heterochromatin [ 11 ]. Besides PML, several other PML-NB components including Sp100, hDaxx, ATRX, and MORC3 function as restriction factors and contribute to the repression of viral gene expression in a cooperative manner [ 12 – 16 ].

To combat viral infections, host organisms have developed an intricate defense network comprising the intrinsic, innate, and adaptive immune response. While innate and adaptive defense mechanisms rely on pathogen-induced activation, the intrinsic immune system is conferred by constitutively expressed restriction factors thus mediating a front-line defense against invading pathogens [ 1 ]. Since the discovery of the first class of restriction factors targeting retroviral capsids, numerous cellular factors have been identified that restrict diverse steps in the life cycle of viruses [ 2 ]. During the evolutionary “arms race”, however, viruses have evolved means to evade or directly counteract these antiviral host factors, mainly by expressing antagonistic proteins. The evolutionary pressure that restriction factors and antagonists have exerted on each other resulted in further adaptations at the virus-host interface. Thus, restriction factors are often less effective against viral infections of their natural host but constitute potent barriers to cross-species infections [ 3 ].

Results

The domain organization of IE1 is conserved across primates and rodents The architecture of cytomegalovirus IE1 proteins appears to be evolutionary conserved across species. An in silico disorder prediction of the rodent member rat cytomegalovirus IE1 protein (ratIE1) is in agreement with the presence of a folded core domain that is flanked by a short partially or fully disordered N-terminal segment as well as a disordered extended C-terminal segment as previously observed in the primate IE1 proteins from human (humIE1) and rhesus (rhesIE1) cytomegalovirus (Fig 1A) [22]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Analysis of the domain organization of ratIE1. (A) In silico disorder prediction analysis of human (hum), rhesus (rhes) and rat (rat) cytomegalovirus IE1 sequences using IUPred2A [69]. The disorder score for all three proteins suggest a globular domain with disordered N- and C-termini (scores ≥ 0.5 indicate disorder). (B) Limited proteolysis of recombinant ratIE1. Purified ratIE1 was incubated with subtilisin (1 mU protease per mg ratIE1) for different times, and samples were analyzed by SDS-PAGE and Coomassie blue staining. (C) CD spectroscopy of humIE1 14–382, ratIE1 1–392 and ratIE1 30–392. The spectra were normalized at 207 nm as suggested by Raussens and coworkers [57]. https://doi.org/10.1371/journal.ppat.1009863.g001 An experimental validation of the in silico prediction via a limited proteolysis digestion of full-length recombinant ratIE1 (residues 1 to 565) yielded a single and stable 45 kDa fragment (Fig 1B). A mass spectrometry analysis of this fragment revealed that it extends from residues 1 to 392 of the ratIE1 sequence (S1 Fig). It includes the very N-terminal residues of ratIE1, which in case of the homologous rhesIE1 and humIE1 proteins, were prone to digestion in previous experiments and therefore postulated as not being part of the core domains in these proteins (humIE1 CORE , residues 14 to 382; rhesIE1 CORE , residues 36 to 395) [22]. Interestingly, the in silico disorder analysis of ratIE1 anticipated this result since the predicted disorder tendency for the first 16 residues is considerably reduced in ratIE1 versus hum- and rhesIE1 (Fig 1A). One humIE1 and two ratIE1 variants were produced for further characterization. The two ratIE1 variants, covering residues 1 to 392 and residues 30 to 392, show an almost identical all-α-helical secondary structure composition as analyzed by CD spectroscopy (Fig 1C). Moreover, the CD spectra of the two ratIE1 variants are almost indistinguishable from that of humIE1 CORE in agreement with the assumption of a shared core domain in IE1 proteins (Fig 1C). Of the two ratIE1 variants, only the N-terminally truncated variant yielded protein crystals. This variant, covering residues 30 to 392, is from here-on referred to as the ratIE1 CORE domain. Taken together, ratIE1, humIE1 and rhesIE1 share an approximately 350-residue, all-α-helical core domain that is flanked by a short, fully or partially disordered region at the N-terminus and a 110- to 170-residue-long disordered region at the C-terminus.

RatIE1, humIE1 and rhesIE1 share a unique fold RatIE1 CORE and humIE1 CORE share a highly similar overall fold, which bears close resemblance to that of the previously determined rhesIE1 CORE structure (Figs 2 and S2) [22]. All three IE1 CORE proteins display a femur-like structure consisting of α-helices only. These are arranged into two head regions interconnected by a stalk region composed of three to four long α-helices. HumIE1 CORE resembles rhesIE1 CORE more closely than ratIE1 CORE . HumIE1 CORE can be superimposed onto rhesIE1 CORE with an rmsd Cα value of 2.3 Å, while the structures of humIE1 CORE and ratIE1 CORE differ by an rmsd Cα value of as high as 4.6 Å (Table 2). The structural deviations between these proteins are paralleled by marked differences in sequence identities. While humIE1 CORE and rhesIE1 CORE can be aligned with 24% sequence identity, the sequence identity between humIE1 CORE and ratIE1 CORE amounts to only 22% (Table 2). PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Sequence and structure similarities between IE1 CORE domains. https://doi.org/10.1371/journal.ppat.1009863.t002 It is known from comparative structural biology that the lower the sequence identities between the proteins, the more dissimilar the structures of the respective proteins are and vice versa [24]. However, sequence identities as low as 24 or 22% fall below the cut-off value of 28% that has been derived as a lower limit for safely inferring structural details and overall similarities from sequence identities in proteins of more than 200 residues in length [25,26]. As a consequence of the low sequence identities, humIE1 CORE , rhesIE1 CORE and ratIE1 CORE exhibit marked differences (Figs 2 and S2). Thus, helix H1 is significantly shorter in ratIE1 CORE , and the position of H2 is rotated by approximately 90° in comparison to humIE1 CORE and rhesIE1 CORE . Furthermore, a kink separates helices H5 and H6 in humIE1 CORE and rhesIE1 CORE , whereas ratIE1 CORE contains one continuous helix termed H5/6. Besides this, the curvature of several helices, namely H3, H6 and H9, also slightly differs between humIE1 CORE and rhesIE1 CORE on one hand and ratIE1 CORE on the other hand. At the same time, ratIE1 CORE has an additional helix H12 at the C-terminus in comparison to humIE1 CORE and rhesIE1 CORE , which consist of eleven helices in total. A DALI search against the entire protein data bank (PDB, performed in February 2021) unambiguously identifies these three proteins as forming a unique structure family (S3 Table) [27,28]. Additional candidate homologous proteins, as identified by DALI, either display excessively high rmsd Cα values exceeding 8 Å when aligning up to 240 residues or the structural homology is limited to considerably smaller segments of about 100 residues in the compared proteins so that rmsd Cα values of about 3 Å upwards are obtained (S3 Table). This shows that clear structural homology extending over the entire length of the compared protein structures is only detectable within the group of IE1 CORE proteins, but not to any other protein of known structure.

CMV IE1 proteins display an identical dimerization mode All IE1 CORE proteins not only display a similar and unique overall fold but also form highly similar dimeric assemblies. In the ratIE1 CORE and humIE1 CORE crystals, the crystallographic asymmetric units contain a single protein chain. However, in both cases, inspection of the crystal packing interactions reveals the presence of tightly interacting dimers (Fig 3A and 3B). In these dimers, the two protomers are related by crystallographic two-fold symmetry axes (Fig 3C) and hence, the dimers display C 2 point group symmetry similarly to previously described rhesIE1 CORE [22]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Shared dimerization mode in ratIE1 CORE , humIE1 CORE and rhesIE1 CORE . (A) Dimers of IE1 CORE proteins are depicted viewing along or perpendicular to the dimerization axis as well as with cylinders placed through all atoms of the respective molecule. (A) ratIE1 CORE , (B) humIE1 CORE and (C) superposition of ratIE1 CORE , humIE1 CORE and rhesIE1 CORE (the latter is taken from PDB entry 4WID) . The dimeric assembly is characterized by a two-fold rotation axis that interrelates the monomers in the dimer (highlighted in panel C). The cylinder representations show that the two monomer axes of least inertia form an angle of about 23° in the dimers. Panel C shows that this angle is highly similar in ratIE1 CORE (24.4°), humIE1 CORE (22.4°) and rhesIE1 CORE (21.2°). https://doi.org/10.1371/journal.ppat.1009863.g003 In all IE1 CORE structures, the monomers dimerize via an identical interface, and highly similar crossing-angles are adopted between monomers (Fig 3C). The cross-species conserved quaternary arrangement is also evident when comparing the superposition of dimers with the superposition of monomers. When superimposing the various dimers, the calculated rmsd Cα values are only marginally higher than the deviations obtained between monomers in support of a conserved quaternary assembly in IE1 CORE proteins (Table 2). Analysis of all IE1 CORE structures with program EPPIC suggests that the dimeric assembly corresponds to the biologically active unit of IE1 CORE [29]. All remaining protein interfaces observed in the various crystals are classified as mere crystal packing contacts. The sizes of the dimer interfaces are also comparable between IE1 CORE proteins ranging from 2240 to 2430 and 2470 Å2 in ratIE1 CORE , humIE1 CORE and rhesIE1 CORE (PDB entry 4WIC), respectively. Interestingly, an interface of 2470 Å2 is only observed in crystals of rhesIE1 CORE before induction of a crystallographic phase transition [30]. A dehydration of rhesIE1 CORE crystals induces a distinct conformational rearrangement in one segment of one protomer of rhesIE1 CORE , and a more extensive dimer interface of about 3070 Å2 is formed [22,30]. Taken together, the IE1 proteins appear to form a distinct class of proteins characterized by a shared unique tertiary fold and quaternary assembly. At the same time, the sequence identities observed between these proteins map these to the so-called “twilight zone”, where inference of structural details from sequence alignments only has to be cautioned [25,26].

The canonical IE1 CORE fold is built from conserved regions of left- and right-handed coiled-coils The all-α-helical fold of IE1 CORE consists of specific left- and right-handed helix pairings that originate from distinct hydrophobic repeat motifs. The N-terminal head region of ratIE1 CORE , humIE1 CORE and rhesIE1 CORE is formed by helices H3, H7 and H8, and these helices form left-handed coiled-coils (Fig 4). The sequences of these helices mainly contain heptad repeats. In these ‘abcdefg’ repeats, hydrophobic residues are displayed at positions a and d and give rise to left-handed helix crossings (Figs 4 and S3) [31]. The central stalk and C-terminal head regions exhibit more uncommon, right-handed coiled-coils due to the presence of hendecad (undecad) ‘abcdefghijk’ repeats with hydrophobic residues at positions a, d and h [31]. However, whereas the stalk and the adjacent C-terminal head region of ratIE1 CORE are formed by continuous right-handed coiled-coils, this segment is interrupted by a region of left-handed coiled-coils in rhesIE1 CORE and humIE1 CORE (Figs 4 and S3). At the stalk-head transition (H3/H4), rhesIE1 CORE and humIE1 CORE display an insertion of two hydrophilic residues, which point towards the solvent and locally distort the helix geometry to form a sharp kink. In contrast, H3 and H4 of ratIE1 CORE are separated by a short unstructured linker (Fig 4). The primate CMV proteins further lack one heptad repeat in the middle of H6. Overall, the three IE1 CORE structures show similar helix-pairing arrangements. At the same time, specific differences exist in the hydrophobic repeat patterns between the primate and the rodent IE1 CORE structures. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Occurrence and distribution of left- and right-handed coiled-coils in rodent and primate IE1 proteins. Ribbon representation of ratIE1 CORE (A), humIE1 CORE (B) and rhesIE1 CORE (C) colored according to the handedness of helix-pairings. Yellow: left-handed coiled-coils. Cyan: right-handed coiled-coils. Magenta: three-residue insertion. https://doi.org/10.1371/journal.ppat.1009863.g004 These repeat patterns appear more conserved than individual amino acids. Program PROMALS3D was used to generate a structure-based multiple sequence alignment of the three IE1 CORE proteins (S3 Fig) [32]. Surprisingly, at nine positions only, amino acid types are conserved across all three proteins. Derivation of pairwise sequence identities from the structure-based alignment reveals that in case of rhesIE1 CORE and humIE1 CORE , the observed sequence identity matches that obtained with standard sequence alignment algorithms (22 versus 24%, respectively, Table 2). However, when comparing the structure-derived sequence identity between ratIE1 CORE and either humIE1 CORE or rhesIE1 CORE , sequence identities as low as 9% are obtained for both comparisons. These are considerably lower than the 22% sequence identities obtained with standard sequence alignment algorithms. Knowledge of the distribution of hydrophobic repeat motifs could help to more reliably model additional IE1-homologous proteins since these distributions are responsible for the topological arrangement of the α-helices in IE1 CORE . To test this, the sequence of the structurally uncharacterized mouse CMV IE1 (murIE1) protein was manually incorporated into the structure-based sequence alignment of rhesIE1, humIE1 and ratIE1 (S3 Fig). The alignment shows that the regions can be readily identified and that these show high similarity to those of the crystallized IE1 proteins. We propose that these conserved repeat patterns can be used to improve the reliability of sequence alignments and the correctness of homology models, in particular in cases, where sequence identities fall within the “twilight zone”.

The mechanism of PML-NB disruption is conserved among primate and rodent cytomegaloviruses Due to the structural conservation of the IE1 core domain, the question arose whether all IE1 homologs use the same molecular mechanism to disrupt the antiviral PML-NBs. To address this issue, several rat PML (ratPML) deletion mutants were generated and analyzed for an interaction with ratIE1 in HEK293T cells (Fig 5A). Co-immunoprecipitation experiments revealed that ratIE1 binds full-length ratPML and, even more efficiently, the truncated ratPML RBCC protein (Fig 5B, lane 3 and 6). A construct encoding an N-terminally extended ratIE1 CORE protein (ratIE1 1–392) was sufficient for this interaction (Fig 5B, lane 2 and 5), which is in accordance with our previous data on human PML (humPML) and humIE1 [22]. Please note that for all cell-based assays, this N-terminally extended IE1 CORE variant was used since the N-terminus has been proposed to harbor the NLS signal [33]. Deletion of the coiled-coil domain from the ratPML RBCC protein (ratPML RB) abolished the interaction with ratIE1 CORE suggesting that ratPML-NBs are targeted through coiled-coil interactions (Fig 5C, lane 3). Proper folding of the ratPML RB fragment was confirmed by CD spectroscopy, which also revealed a shared secondary structure composition with the corresponding humPML RB construct (S4 Fig). Moreover, we found that ratPML constructs lacking the RING domain (ratPML BCC and ratPML ΔR) are also not able to bind ratIE1 CORE (Fig 5C, lane 4 and 5). However, comparatively low expression levels of such constructs in lysate and precipitation samples hint to a possible requirement of the RING domain for proper folding and solubility of ratPML. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Interaction of ratIE1 with ratPML followed by ratPML deSUMOylation and dispersion. (A) Schematic overview of full-length ratPML and deletion mutants. (B) Efficient interaction of ratIE1 CORE with ratPML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids encoding FLAG-tagged ratIE1 or ratIE1core (residues 1–392) and Myc-tagged ratPML variants. After cell lysis, immunoprecipitation was performed with an anti-FLAG antibody. Co-precipitated ratPML proteins (IP), precipitated ratIE1 proteins, and proteins within the cell lysate (input) were analyzed by Western blotting as indicated. (C) Binding of ratIE1 CORE to ratPML requires both the coiled-coil and the RING domain. HEK293T cells were co-transfected with expression plasmids encoding FLAG-tagged ratPML variants and Myc-tagged ratIE1 CORE (residues 1–382) as indicated. Upper two panels: Western blot detection of ratIE1 and ratPML after immunoprecipitation using an anti-FLAG antibody. Lower two panels: detection of ratIE1 and ratPML in cell lysates before precipitation (input). (D) Inhibition of ratPML SUMOylation by ratIE1 expression. HEK293T cells were transfected with expression plasmids encoding Myc-ratPML, HA-SUMO2 and FLAG-ratIE1 as indicated. After cell harvest, ratPML and SUMOylated ratPML were visualized by Western blotting using anti-Myc and anti-HA antibodies, respectively. Expression of IE1 was analyzed with an anti-FLAG antibody and β-actin was included as internal control. (E) Impact of RCMV infection on ratPML SUMOylation. Rat embryonic fibroblast (REF) cells were infected with RCMV at an MOI of 1.5 or mock infected, and were harvested at indicated times for Western Blot analysis of ratPML (upper panel), ratIE1 (middle panel), and β-actin (lower panel) as loading control. (F) Impact of RCMV infection on ratPML-NB integrity. REF cells were infected with RCMV at an MOI of 0.7 or mock infected, and were harvested at indicated times for immunofluorescence analysis of ratIE1 (left panel) or ratPML (right panel). Cell nuclei were stained with DAPI. F, FLAG; M, Myc; R, RING domain; B, B-boxes; CC, coiled-coil domain. https://doi.org/10.1371/journal.ppat.1009863.g005 Next, we examined whether ratIE1 induces a loss of ratPML SUMOylation and disruption of ratPML foci. Transfection experiments using HEK293T cells showed that ratIE1 is sufficient to induce a loss of SUMOylated ratPML (Fig 5D). To verify this result in the context of infection, rat embryonic fibroblast (REF) cells were either not infected (mock) or infected with RCMV and were analyzed for the SUMOylation state and localization of ratPML at immediate-early times. While SUMOylated forms of ratPML were still detectable at 4 hours post-infection (hpi) and showed comparable levels as in non-infected cells, we observed a clear loss of ratPML SUMOylation beginning at 8 hpi (Fig 5E). In parallel with the depletion of SUMOylated ratPML, the intracellular localization of ratPML and ratIE1 changed from a dot-like to a nuclear diffuse staining pattern (Fig 5F). Since these data match previous findings on rhesIE1 and murIE1, which also abrogate PML SUMOylation and induce a dispersion of PML, we conclude that the molecular mechanism underlying PML-NB disruption is conserved across cytomegalovirus species and relies on the unusual fold of the IE1 core domain [22,34].

PML-NBs are not disrupted during cross-species infection Due to the structural similarity of primate and rat CMV IE1, we next investigated whether IE1 proteins can counteract the PML-based defense during cross-species infection. As shown in Fig 6A, we found that HCMV is capable of entering REF cells and initiate humIE1 expression. However, humIE1 did not localize to nuclear foci, but was distributed throughout the nucleus and did not affect the integrity of ratPML-NBs. In line with this observation, no interaction of humIE1 CORE with ratPML RBCC was detected in co-immunoprecipitation analysis (Fig 6B, lane 3), suggesting that humIE1 is neither able to bind nor to disrupt PML-NBs in rat cells. In a vice versa experiment, we infected primary human fibroblast (HFF) cells with RCMV. We observed no colocalization of ratIE1 with humPML at 4 h after RCMV infection, suggesting that it does not target PML-NBs in human cells (Fig 6C). At later stages, however, ratIE1 was recruited to large, nuclear domains resembling viral pre-replication compartments. Since PML-NBs were found adjacent to but not colocalizing with these structures (Fig 6C, panel 4) and since no interaction of ratIE1 CORE with humPML could be detected (Fig 6D, lane 2), it can be assumed that not humPML but another cellular or viral protein is responsible for recruiting ratIE1 into nuclear domains. Taken together, these data suggest that PML-NBs are not disrupted by IE1 upon cross species infection and point to a contribution of the PML-based intrinsic defense to the species barrier. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Species-specific disruption of PML-NBs during CMV infection. (A) Analysis of PML-NB integrity in rat fibroblasts after HCMV infection. REF cells were infected with HCMV strain AD169 (MOI = 0.5) or mock infected. Cells were harvested at indicated times after infection to analyze the subcellular localization of ratPML (left panel) and humIE1 (right panel). Cell nuclei were stained with DAPI. (B) Species-specific binding of IE1 proteins to ratPML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids coding for the TRIM motif of ratPML fused to a myc-tag (ratPML RBCC) and either FLAG-ratIE1 CORE (residues 1–392), FLAG-humIE1 CORE (residues 1–382) or an empty plasmid (pcDNA3). Afterwards, immunoprecipitation was performed with an anti-FLAG antibody. Left panels: Western blot detection of precipitated IE1 proteins and co-precipitated ratPML RBCC (IP). Right panels: detection of IE1 proteins and ratPML RBCC in cell lysates before precipitation (input). (C) Analysis of PML-NB integrity in human fibroblasts after RCMV infection. HFF cells were infected with RCMV-E (MOI = 0.5) or mock infected. Cells were fixed at indicated times for immunofluorescence analysis of humPML and ratIE1. Cell nuclei were visualized by DAPI staining. (D) Species-specific binding of IE1 proteins to humPML in co-immunoprecipitation analysis. HEK293T cells were co-transfected with expression plasmids encoding myc-tagged humPML and either FLAG-ratIE1 CORE (residues 1–392), FLAG-humIE1 CORE (residues 1–382) or an empty plasmid (pcDNA3). After immunoprecipitation of IE1 with an anti-FLAG antibody, co-precipitated humPML (left panels) as well as proteins in the lysate before precipitation (right panels) were detected by Western blotting. https://doi.org/10.1371/journal.ppat.1009863.g006

IE1 induces PML-NB dispersal in a species-specific manner In order to analyze the cross-species activity of IE1 homologs in absence of other viral proteins, we performed a set of experiments using transduced fibroblasts. Lentiviral vectors were utilized to establish human fibroblast (HFF) and rat fibroblast (REF) cells with doxycycline-inducible expression of FLAG-tagged humIE1 or FLAG-tagged ratIE1 as well as control cells. Subsequent immunofluorescence analysis of HFF cell populations in absence or presence of doxycycline revealed a clear dispersal of PML foci upon humIE1 expression (Fig 7A, panel 4), whereas ratIE1 did neither colocalize with nor disrupt PML-NBs (Fig 7A, panel 6). Quantification of PML foci per cell nucleus corroborated this finding by showing a sharp decline of PML foci in doxycycline-treated HFF/humIE1, while induction of ratIE1 expression did not alter the number of PML-NBs (Fig 7B). In accordance, we observed that humIE1, but not ratIE1, is able to inhibit the SUMOylation of PML in HFF cells (Fig 7C). Equivalent results were obtained in REF cells since only expression of ratIE1 and not humIE1 resulted in dispersal of PML foci (Fig 7D and 7E) and loss of PML SUMOylation (Fig 7F). Overall, our data suggest that the slight structural differences observed in the core domain of primate and rodent IE1 proteins represent evolutionary adaptations to the respective host and result in species-specific targeting of PML-NBs. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 7. Species-specific disruption of PML-NBs in cells stably expressing IE1. (A, B) Effect of humIE1 and ratIE1 on the integrity of PML foci in human fibroblasts. Human fibroblasts with doxycycline-inducible expression of FLAG-tagged humIE1 (HFF/humIE1), FLAG-tagged ratIE1 (HFF/ratIE1) or control cells (HFF/control) were either left untreated (- Dox) or were treated with doxycycline (+ Dox) for 24 h. The cells were fixed for immunofluorescence staining of endogenous humPML and of IE1 proteins using an anti-FLAG antibody (A), followed by quantitation of humPML foci numbers in 50 cell nuclei per sample (B). (C) Impact of humIE1 and ratIE1 on the SUMOylation state of humPML. HFF/humIE1, HFF/ratIE1 or control cells were either left untreated (- Dox) or were treated with doxycycline (+ Dox). 24 h later, cells were harvested for Western Blot detection of IE1 proteins using an anti-FLAG antibody (upper panel), humPML (middle panel), and β-actin as loading control (lower panel). (D, E) Effect of humIE1 and ratIE1 on the integrity of PML foci in rat fibroblasts. Rat fibroblasts with doxycycline-inducible expression of FLAG-tagged humIE1 (REF/humIE1), FLAG-tagged rIE1 (REF/ratIE1) or control cells (REF/control) were either mock treated (- Dox) or were treated with doxycycline (+ Dox) for 24 h. The cells were fixed for immunofluorescence staining of endogenous ratPML and for IE1 proteins using an anti-FLAG antibody (D), followed by quantitation of ratPML foci numbers in 50 cell nuclei per sample (E). (F) Impact of humIE1 and ratIE1 on the SUMOylation state of ratPML. REF/humIE1, REF/ratIE1 or control REF were either left untreated (- Dox) or were treated with doxycycline (+ Dox). 24 h later, cells were harvested for Western Blot detection of IE1 proteins using an anti-FLAG antibody (upper panel), ratPML (middle panel), and β-actin as loading control (lower panel). https://doi.org/10.1371/journal.ppat.1009863.g007

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1009863

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