Laurent Dianoux

Resistance to Rabies Virus Infection Conferred by the PMLIV Isoform

ABSTRACT Various reports implicate PML and PML nuclear bodies (NBs) in an intrinsic antiviral response targeting diverse cytoplasmic replicating RNA viruses. PML conjugation to the small ubiquitin-like modifier (SUMO) is required for its localization within NBs. PML displays antiviral effects in vivo, as PML deficiency renders mice more susceptible to infection with the rhabdovirus vesicular stomatitis virus (VSV). Cells derived from these mice are also more sensitive to infection with rabies virus, another member of the rhabdovirus family. Alternative splicing from a single gene results in the synthesis of several PML isoforms, and these are classified into seven groups, designated PMLI to -VII. We report here that expression of PMLIV or PMLIVa, which is missing exon 5, inhibited viral mRNA and protein synthesis, leading to a reduction in viral replication. However, the expression of other nuclear isoforms (PMLI to -VI) and cytoplasmic PMLVIIb failed to impair viral production. This antiviral effect required PMLIV SUMOylation, as it was not observed with PMLIV 3KR, in which the lysines involved in SUMO conjugation were mutated. Thus, PMLIV and PMLIVa may exert this isoform-specific function through interaction with specific NB protein partners via their common C-terminal region.

Role of SUMO in RNF4-mediated Promyelocytic Leukemia Protein (PML) Degradation Sumoylation of pml and phospho-switch control of its sumo binding domain dissected in living cells

Promyelocytic leukemia protein (PML) is a tumor suppressor acting as the organizer of subnuclear structures called PML nuclear bodies (NBs). Both covalent modification of PML by the small ubiquitin-like modifier (SUMO) and non-covalent binding of SUMO to the PML SUMO binding domain (SBD) are necessary for PML NB formation and maturation. PML sumoylation and proteasome-dependent degradation induced by the E3 ubiquitin ligase, RNF4, are enhanced by the acute promyelocytic leukemia therapeutic agent, arsenic trioxide (As2O3). Here, we established a novel bioluminescence resonance energy transfer (BRET) assay to dissect and monitor PML/SUMO interactions dynamically in living cells upon addition of therapeutic agents. Using this sensitive and quantitative SUMO BRET assay that distinguishes PML sumoylation from SBD-mediated PML/SUMO non-covalent interactions, we probed the respective roles of covalent and non-covalent PML/SUMO interactions in PML degradation and interaction with RNF4. We found that, although dispensable for As2O3-enhanced PML sumoylation and RNF4 interaction, PML SBD core sequence was required for As2O3- and RNF4-induced PML degradation. As confirmed with a phosphomimetic mutant, phosphorylation of a stretch of serine residues, contained within PML SBD was needed for PML interaction with SUMO-modified protein partners and thus for NB maturation. However, mutation of these serine residues did not impair As2O3- and RNF4-induced PML degradation, contrasting with the known role of these phosphoserine residues for casein kinase 2-promoted PML degradation. Altogether, these data suggest a model whereby sumoylation- and SBD-dependent PML oligomerization within NBs is sufficient for RNF4-mediated PML degradation and does not require the phosphorylation-dependent association of PML with other sumoylated partners.

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 PMLIII-expressing 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 SUMO-interacting motif of PML. Together, these findings reveal a new mechanism evolved by EMCV to antagonize the PML pathway in the interferon-induced antiviral defense.

Requirement of PML SUMO interacting motif for RNF4- or arsenic trioxide-induced degradation of nuclear PML isoforms.

PML, the organizer of nuclear bodies (NBs), is expressed in several isoforms designated PMLI to VII which differ in their C-terminal region due to alternative splicing of a single gene. This variability is important for the function of the different PML isoforms. PML NB formation requires the covalent linkage of SUMO to PML. Arsenic trioxide (As2O3) enhances PML SUMOylation leading to an increase in PML NB size and promotes its interaction with RNF4, a poly-SUMO-dependent ubiquitin E3 ligase responsible for proteasome-mediated PML degradation. Furthermore, the presence of a bona fide SUMO Interacting Motif (SIM) within the C-terminal region of PML seems to be required for recruitment of other SUMOylated proteins within PML NBs. This motif is present in all PML isoforms, except in the nuclear PMLVI and in the cytoplasmic PMLVII. Using a bioluminescence resonance energy transfer (BRET) assay in living cells, we found that As2O3 enhanced the SUMOylation and interaction with RNF4 of nuclear PML isoforms (I to VI). In addition, among the nuclear PML isoforms, only the one lacking the SIM sequence, PMLVI, was resistant to As2O3-induced PML degradation. Similarly, mutation of the SIM in PMLIII abrogated its sensitivity to As2O3-induced degradation. PMLVI and PMLIII-SIM mutant still interacted with RNF4. However, their resistance to the degradation process was due to their inability to be polyubiquitinated and to recruit efficiently the 20S core and the β regulatory subunit of the 11S complex of the proteasome in PML NBs. Such resistance of PMLVI to As2O3-induced degradation was alleviated by overexpression of RNF4. Our results demonstrate that the SIM of PML is dispensable for PML SUMOylation and interaction with RNF4 but is required for efficient PML ubiquitination, recruitment of proteasome components within NBs and proteasome-dependent degradation of PML in response to As2O3.

Small Ubiquitin-like Modifier Alters IFN Response.

IFNs orchestrate immune defense through induction of hundreds of genes. Small ubiquitin-like modifier (SUMO) is involved in various cellular functions, but little is known about its role in IFN responses. Prior work identified STAT1 SUMOylation as an important mode of regulation of IFN-γ signaling. In this study, we investigated the roles of SUMO in IFN signaling, gene expression, protein stability, and IFN-induced biological responses. We first show that SUMO overexpression leads to STAT1 SUMOylation and to a decrease in IFN-induced STAT1 phosphorylation. Interestingly, IFNs exert a negative retrocontrol on their own signaling by enhancing STAT1 SUMOylation. Furthermore, we show that expression of each SUMO paralog inhibits IFN-γ–induced transcription without affecting that of IFN-α. Further, we focused on IFN-induced gene products associated to promyelocytic leukemia (PML) nuclear bodies, and we show that neither IFN-α nor IFN-γ could increase PML and Sp100 protein expression because they enhanced their SUMO3 conjugation and subsequent proteasomal degradation. Because it is known that SUMO3 is important for the recruitment of RING finger protein 4, a poly–SUMO-dependent E3 ubiquitin ligase, and that PML acts as a positive regulator of IFN-induced STAT1 phosphorylation, we went on to show that RING finger protein 4 depletion stabilizes PML and is correlated with a positive regulation of IFN signaling. Importantly, inhibition of IFN signaling by SUMO is associated with a reduction of IFN-induced apoptosis, cell growth inhibition, antiviral defense, and chemotaxis. Conversely, inhibition of SUMOylation results in higher IFN-γ–induced STAT1 phosphorylation and biological responses. Altogether, our results uncover a new role for SUMO in the modulation of IFN response.

In vitro induction of early mouse embryo intracisternal particles (ε particles) in cultured cell lines

Summary— The experimental induction of ε particles, retrovirus‐like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent‐cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycine, deoxy‐fluorouracile or 5‐azacytine. However, only two mouse‐derived cell lines: KiBALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed ε particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previously shown to attenuate IAP production, had no effect on that of ε particles.

Differential effects of SUMO1 and SUMO3 on PKR activation and stability

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a serine/threonine kinase that exerts its own phosphorylation and the phosphorylation of the α subunit of the protein synthesis initiation factor eIF-2α. PKR was identified as a target of SUMOylation and the triple PKR-SUMO deficient mutant on Lysine residues K60-K150-K440 has reduced PKR activity. We report that SUMO1 and SUMO3 expression exert differential effects on PKR localization, activation and stability. SUMO1 or SUMO3 did not alter the repartition of PKR in the cytoplasm and the nucleus. However, in SUMO3-expressing cells PKR was found more concentrated around the perinuclear membrane and was recruited from small speckles to nuclear dots. Interestingly, SUMO1 expression alone resulted in PKR and eIF-2α activation, whereas SUMO3 reduced PKR and eIF-2α activation upon viral infection or dsRNA transfection. In addition, encephalomyocarditis virus (EMCV) enhanced PKR conjugation to SUMO1 and SUMO3 but only SUMO3 expression promoted caspase-dependent EMCV-induced PKR degradation. Furthermore, the higher EMCV-induced PKR activation by SUMO1 was correlated with an inhibition of EMCV. Importantly SUMO1, by inducing PKR activation in the absence of viral infection, and SUMO3, by counteracting both PKR activation and stability upon viral infection, shed a new light on the differential effects of SUMO-modified PKR.

Les mécanismes de l’action antivirale des interférons : la voie PML

Decouverts depuis 1957 pour leurs proprietes antivirales, les interferons (IFN) sont une famille de cytokines qui ne cesse de s’agrandir avec des activites biologiques de plus en plus diversifiees, telles que les activites antitumorale et immunoregulatrice. Les IFN sont classes en trois types I, II et III. Ils se fixent a differents recepteurs cellulaires specifiques et induisent, via la voie Jak/Stat, l’expression de plus de trois cents genes dont les produits sont responsables de leurs effets biologiques. Certains ont ete impliques dans le mecanisme d’action antivirale comme la proteine kinase dependante de l’ARN double brin (PKR), la 2’5’oligoadenylate synthetase/RNaseL, et les proteines Mx. Cependant, les cellules triplement deficientes pour ces trois voies (PKR, RNaseL et Mx) presentent toujours une protection par l’IFN contre l’infection virale. Il existe donc d’autres voies antivirales. L’une d’elles implique la proteine PML (promyelocytic leukemia). Cette revue resume les proprietes des proteines mediatrices des effets antiviraux de l’IFN avec une attention particuliere pour la proteine PML ainsi que les strategies developpees par des virus de familles differentes pour contrecarrer cette defense.