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

Abstract

PML nuclear bodies (PML-NBs) are enigmatic structures of the cell nucleus that act as key mediators of intrinsic immunity against viral pathogens. PML itself is a member of the E3-ligase TRIM family of proteins that regulates a variety of innate immune signaling pathways. Consequently, viruses have evolved effector proteins to modify PML-NBs; however, little is known concerning structure-function relationships of viral antagonists. The herpesvirus human cytomegalovirus (HCMV) expresses the abundant immediate-early protein IE1 that colocalizes with PML-NBs and induces their dispersal, which correlates with the antagonization of NB-mediated intrinsic immunity. Here, we delineate the molecular basis for this antagonization by presenting the first crystal structure for the evolutionary conserved primate cytomegalovirus IE1 proteins. We show that IE1 consists of a globular core (IE1 _CORE) flanked by intrinsically disordered regions. The 2.3 Å crystal structure of IE1 _CORE displays an all α-helical, femur-shaped fold, which lacks overall fold similarity with known protein structures, but shares secondary structure features recently observed in the coiled-coil domain of TRIM proteins. Yeast two-hybrid and coimmunoprecipitation experiments demonstrate that IE1 _CORE binds efficiently to the TRIM family member PML, and is able to induce PML deSUMOylation. Intriguingly, this results in the release of NB-associated proteins into the nucleoplasm, but not of PML itself. Importantly, we show that PML deSUMOylation by IE1 _CORE is sufficient to antagonize PML-NB-instituted intrinsic immunity. Moreover, co-immunoprecipitation experiments demonstrate that IE1 _CORE binds via the coiled-coil domain to PML and also interacts with TRIM5α We propose that IE1 _CORE sequesters PML and possibly other TRIM family members via structural mimicry using an extended binding surface formed by the coiled-coil region. This mode of interaction might render the antagonizing activity less susceptible to mutational escape.

Author Summary

Research of the last few years has revealed that microbial infections are not only controlled by innate and adaptive immune mechanisms, but also by cellular restriction factors, which give cells the capacity to resist pathogens. PML nuclear bodies (PML-NBs) are dot-like nuclear structures representing multiprotein complexes that consist of the PML protein, a member of the TRIM family of proteins, as well as a multitude of additional regulatory factors. PML-NB components act as a barrier against many viral infections; however, viral antagonistic proteins have evolved to modify PML-NBs, thus abrogating this cellular defense. Here, we delineate the molecular basis for antagonization by the immediate-early protein IE1 of the herpesvirus human cytomegalovirus. We present the first crystal structure for the evolutionary conserved core domain (IE1 _CORE) of primate cytomegalovirus IE1, which exhibits a novel, unusual fold. IE1 _CORE modifies PML-NBs by releasing other PML-NB proteins into the nucleoplasm which is sufficient to antagonize intrinsic immunity. Importantly, IE1 _CORE shares secondary structure features with the coiled-coil domain (CC) of TRIM factors, and we demonstrate strong binding of IE1 to the PML-CC. We propose that IE1 _CORE sequesters PML and possibly other TRIM family members via an extended binding surface formed by the coiled-coil domain.