SUMOylation Promotes PML Degradation during Encephalomyocarditis Virus Infection

Abstract

The promyelocytic leukemia (PML) protein is expressed in the diffuse nuclear fraction of the nucleoplasm and in matrix-associated structures, known as nuclear bodies (NBs). PML NB formation requires the covalent modification of PML to SUMO. The noncovalent interactions of SUMO with PML based on the identification of a SUMO-interacting motif within PML seem to be required for further recruitment within PML NBs of SUMOylated proteins. RNA viruses whose replication takes place in the cytoplasm and is inhibited by PML have developed various strategies to counteract the antiviral defense mediated by PML NBs. We show here that primary fibroblasts derived from PML knockout mice are more sensitive to infection with encephalomyocarditis virus (EMCV), suggesting that the absence of PML results in an increase in EMCV replication. Also, we found that EMCV induces a decrease in PML protein levels both in interferon-treated cells and in PMLIIIexpressing cells. Reduction of PML was carried out by the EMCV 3C protease. Indeed, at early times postinfection, EMCV induced PML transfer from the nucleoplasm to the nuclear matrix and PML conjugation to SUMO-1, SUMO-2, and SUMO-3, leading to an increase in PML body size where the viral protease 3C and the proteasome component were found colocalizing with PML within the NBs. This process was followed by PML degradation occurring in a proteasome-and SUMO-dependent manner and did not involve the SUMOinteracting motif of PML. Together, these findings reveal a new mechanism evolved by EMCV to antagonize the PML pathway in the interferon-induced antiviral defense.The PML (promyelocytic leukemia) gene was originally identified through its fusion with the RAR␣ gene in the t(15;17) translocation found in acute promyelocytic leukemia (APL) (14). PML, also known as TRIM19, is expressed in the diffuse nuclear fraction of the nucleoplasm and in matrixassociated structures, known as nuclear bodies (NBs) (17, 30). PML functions as the organizer of PML NBs, which are dynamic structures harboring numerous transiently and permanently localized proteins (35). The RBCC/TRIM motif, which contains a C 3 HC 4 (RING finger) zinc-binding domain, two cysteine/histidine-rich motifs (the B boxes B1 and B2), an a helical coiled-coil region (RBCC), is embedded within the PML protein and is required for PML NB formation (28). Due to alternative splicing, many PML isoforms are synthesized, and they are classified into seven groups, designated PML I to PML VII (reviewed in reference 28). They share the N-terminal region (exons 1 to 3), which encodes the RBCC motif, whereas they differ in their C-terminal regions. Posttranslational modification of PML by SUMO (small ubiquitin-like modifier), a ubiquitin-like protein of 11 kDa, is another requirement for PML NB formation. SUMO is covalently coupled to PML through its lysines 65, 160, and 490 via a process called SUMOylation (29). The noncovalent interaction of SUMO with PML through a SUMO-interacting motif (SIM; also named SBD for SUMO binding domain) (46) has been sug...

Promyelocytic Leukemia Isoform IV Confers Resistance to Encephalomyocarditis Virus via the Sequestration of 3D Polymerase in Nuclear Bodies

“…Mouse embryonic fibroblasts (MEFs) derived from knockout PML mice are more sensitive than MEFs from wildtype mice to EMCV infection (12). To determine whether particular PML isoforms inhibit EMCV production, we stably expressed each of the different PML isoforms (PMLI to PMLVII) in U373MG cells.…”

“…PML knockout mice are more sensitive than wild-type mice are to lymphocytic choriomeningitis virus (LCMV) and VSV infections (4), confirming the antiviral effect of PML in vivo. Also, cells are more sensitive to encephalomyocarditis virus (EMCV) infection in the absence of PML (12).…”

The SP100 component of ND10 enhances accumulation of PML and suppresses replication and the assembly of HSV replication compartments

“…The response of other viruses varies. In encephalomyocarditis virus-infected cells, PML is degraded by the viral protease 3C (16). Human cytomegalovirus redistributes PML and disrupts the ND10 bodies (17,18).…”

Implication des corps nucléaires PML dans l’immunité intrinsèque et innée

“…Bien que l'EMCV se réplique entièrement dans le cytoplasme, la protéine virale 3D polymérase (3Dpol) et la protéase virale 3Cpro se retrouvent dans le noyau au début de l'infection, formant des structures ponctuées [17,31]. Les fibroblastes embryonnaires qui dérivent des souris invalidées pour le gène pml (MEF PML -/-) sont plus sensibles à l'infection par l'EMCV que les MEF parentaux [32], suggérant l'implication de PML dans la défense contre ce virus. Une étude surexprimant chacune des isoformes de PML (PMLI à PMLVIIb) d'une manière stable dans des cellules humaines a montré que seule PMLIV protège les cellules de l'infection par L'EMCV.…”