Crystal Structure of Cytomegalovirus IE1 Protein Reveals Targeting of TRIM Family Member PML via Coiled-Coil Interactions

Scherer, Myriam; Klingl, Stefan; Sevvana, Madhumati; Otto, Victoria; Schilling, Eva-Maria; Stump, Joachim D.; Müller, Regina; Reuter, Nina; Sticht, Heinrich; Muller, Yves A.; Stamminger, Thomas

doi:10.1371/journal.ppat.1004512

Cited by 58 publications

(120 citation statements)

References 87 publications

(117 reference statements)

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“…6D). This finding is additionally supported by structural analysis of IE1 CORE , which provides no evidence for the presence of a potential active site with hydrolase activity (30).…”

Section: Discussionmentioning

confidence: 89%

“…Having shown that lysine 160 is selectively affected by HCMV IE1, the question arose whether IE1 can disturb this As 2 O 3 -mediated degradation of PML. To clarify this issue, we utilized HFF cells with doxycycline-inducible expression of wild-type IE1 or truncated IE1 1-382 (IE1 CORE ), which was described to be sufficient for loss of PML SUMOylation (30,42). As illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For PML, the coiled-coil-mediated dimerization is thought to be a prerequisite for its SUMO E3 ligase activity and for PML autoSUMOylation. Since IE1 CORE was shown to interact with the coiled-coil domain of PML, we asked whether IE1 affects PML SUMOylation by disrupting the coiled-coil-mediated self-interaction (30). For this purpose, we performed competitive coimmunoprecipitation experiments using two differentially tagged versions of PML and increasing amounts of IE1 CORE .…”

Section: Ie1 Prevents Pml De Novo Sumoylation In Vivomentioning

confidence: 99%

“…The crystal structure further revealed a similarity between the crystal structure of IE1 CORE and that of TRIM coiled-coil domains. Interestingly, experiments from our group and others demonstrated that IE1 targets the coiled-coil domain of PML and also interacts with other members of this protein family, such as TRIM5␣ and TRIM33 (30,39). This led the authors to propose a model whereby IE1 targets PML via an extended binding surface which involves coiled-coil interactions, thereby interfering with PML SUMOylation (30).…”

mentioning

confidence: 98%

“…Previous studies identified a central large core region of IE1 as being sufficient for interaction with PML; however, further attempts to narrow down the binding region by using mutations and deletions failed (29,30,(34)(35)(36)(37)(38). Indeed, the crystal structure of the rhesus cytomegalovirus IE1 CORE , which was recently solved by our group, provided strong evidence that IE1 CORE consists of a single contiguous domain which is very sensitive to deletion mutagenesis (30). The crystal structure further revealed a similarity between the crystal structure of IE1 CORE and that of TRIM coiled-coil domains.…”

mentioning

confidence: 99%

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

Schilling

Scherer

Reuter

et al. 2017

J Virol

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PML nuclear bodies (NBs) are accumulations of cellular proteins embedded in a scaffold-like structure built by SUMO-modified PML/TRIM19. PML and other NB proteins act as cellular restriction factors against human cytomegalovirus (HCMV); however, this intrinsic defense is counteracted by the immediate early protein 1 (IE1) of HCMV. IE1 directly interacts with the PML coiled-coil domain via its globular core region and disrupts NB foci by inducing a loss of PML SUMOylation. Here, we demonstrate that IE1 acts via abrogating the de novo SUMOylation of PML. In order to overcome reversible SUMOylation dynamics, we made use of a cell-based assay that combines inducible IE1 expression with a SUMO mutant resistant to SUMO proteases. Interestingly, we observed that IE1 expression did not affect preSUMOylated PML; however, it clearly prevented de novo SUMO conjugation. Consistent results were obtained by in vitro SUMOylation assays, demonstrating that IE1 alone is sufficient for this effect. Furthermore, IE1 acts in a selective manner, since K160 was identified as the main target lysine. This is strengthened by the fact that IE1 also prevents As 2 O 3 -mediated hyperSUMOylation of K160, thereby blocking PML degradation. Since IE1 did not interfere with coiled-coil-mediated PML dimerization, we propose that IE1 affects PML autoSUMOylation either by directly abrogating PML E3 ligase function or by preventing access to SUMO sites. Thus, our data suggest a novel mechanism for how a viral protein counteracts a cellular restriction factor by selectively preventing the de novo SUMOylation at specific lysine residues without affecting global protein SUMOylation. IMPORTANCEThe human cytomegalovirus IE1 protein acts as an important antagonist of a cellular restriction mechanism that is mediated by subnuclear structures termed PML nuclear bodies. This function of IE1 is required for efficient viral replication and thus constitutes a potential target for antiviral strategies. In this paper, we further elucidate the molecular mechanism for how IE1 antagonizes PML NBs. We show that tight binding of IE1 to PML interferes with the de novo SUMOylation of a distinct lysine residue that is also the target of stress-mediated hyperSUMOylation of PML. This is of importance since it represents a novel mechanism used by a viral antagonist of intrinsic immunity. Furthermore, it highlights the possibility of developing small molecules that specifically abrogate this PML-antagonistic activity of IE1 and thus inhibit viral replication.KEYWORDS PML nuclear bodies, human cytomegalovirus, immediate early 1, intrinsic immunity, nuclear domain 10, sumoylation P romyelocytic leukemia protein (PML) is known to act as organizer of subnuclear, matrix-associated structures which can be detected as spherical dots of approximately 0.1 to 1.0 m (1). These structures, termed PML nuclear bodies (NBs) or nuclear domain 10 (ND10), are multiprotein complexes consisting of more than 160 proteins.

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“…6D). This finding is additionally supported by structural analysis of IE1 CORE , which provides no evidence for the presence of a potential active site with hydrolase activity (30).…”

Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Section: Ie1 Prevents Pml De Novo Sumoylation In Vivomentioning

confidence: 99%

mentioning

confidence: 98%