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

Abstract

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.

The ND10 Component Promyelocytic Leukemia Protein Acts as an E3 Ligase for SUMOylation of the Major Immediate Early Protein IE1 of Human Cytomegalovirus

“…Nevertheless, one might speculate that PML has the potential to negatively influence the activity of viral regulatory proteins by modulating their posttranslational modification status. This, however, may be abrogated by the propensity of IE1 to block PML E3 ligase functions (25).…”

“…In the case of the betaherpesvirus human cytomegalovirus (HCMV), we and others have shown that the ND10-instituted antiviral state has to be antagonized by the virus in order to efficiently start the viral gene expression program for lytic replication (reviewed in references 20 and 21). In this regard, the HCMV immediate early protein IE1p72 (IE1) plays an important role, as it is able to induce the destruction of this subnuclear structure by abrogating the SUMOylation of PML and Sp100 and it was recently shown that this is due to a blockage of de novo SUMOylation (22)(23)(24)(25). Since covalent as well as noncovalent SUMO interactions constitute the basis for ND10 formation, this ultimately leads to a loss of PML-NB integrity (26)(27)(28)(29).…”

Role of PML-Nuclear Bodies in Human Herpesvirus 6A and 6B Genome Integration

“…In addition to their roles in telomere stability, PML-NBs have antiviral defense functions (36), (35). In contrast to many other viruses including HSV, CMV, EBV and HHV-8, HHV-6A/B infection does not lead to the dispersal of PML-NBs but rather to PML-NBs coalescence (38), (66), (67), (68), (33). Whether this affects antiviral functions of PML-NBs remains to be determined.…”

Feedback-mediated signal conversion promotes viral fitness

“…Interestingly, the mechanisms of action by which pp71 and IE1 antagonize silencing from the PML bodies are different: pp71 induces SUMOyilation and degradation of DAXX (26), while IE1 blocks PML SUMOylation, leading to dispersion of the PML bodies (43).…”