No abstract

Acute promyelocytic leukemia (APL) is associated with the t(15;17) translocation, which generates a PML͞RAR␣ fusion protein between PML, a growth suppressor localized on nuclear matrix-associated bodies, and RAR␣, a nuclear receptor for retinoic acid (RA). PML͞RAR␣ was proposed to block myeloid differentiation through inhibition of nuclear receptor response, as does a dominant negative RAR␣ mutant. In addition, in APL cells, PML͞RAR␣ displaces PML and other nuclear body (NB) antigens onto nuclear microspeckles, likely resulting in the loss of PML and͞or NB functions. RA leads to clinical remissions through induction of terminal differentiation, for which the respective contributions of RAR␣ (or PML͞RAR␣) activation, PML͞ RAR␣ degradation, and restoration of NB antigens localization are poorly determined. Arsenic trioxide also leads to remissions in APL patients, presumably through induction of apoptosis. We demonstrate that in non-APL cells, arsenic recruits the nucleoplasmic form of several NB antigens onto NB, but induces the degradation of PML only, identifying a powerful tool to approach NB function. In APL cells, arsenic targets PML and PML͞RAR␣ onto NB and induces their degradation. Thus, RA and arsenic target RAR␣ and PML, respectively, but both induce the degradation of the PML͞ RAR␣ fusion protein, which should contribute to their therapeutic effects. The difference in the cellular events triggered by these two agents likely stems from RA-induced transcriptional activation and arsenic effects on NB proteins.

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia͞retinoic acid receptor ␣ (PML͞RAR␣) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML͞RAR␣ cleavage, RA triggers degradation of both PML͞RAR␣ and RAR␣. Similarly, in non-APL cells, RA directly targeted RAR␣ and RAR␣ fusions to the proteasome degradation pathway. Activation of either RAR␣ or RXR␣ by specific agonists induced degradation of both proteins. Conversely, a mutation in RAR␣ that abolishes heterodimer formation and DNA binding, blocked both RAR␣ and RXR␣ degradation. Mutations in the RAR␣ DNA-binding domain or AF-2 transcriptional activation region also impaired RAR␣ catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.

The PML protein is associated to nuclear bodies (NBs) whose functions are as yet unknown. PML and two other NBs-associated proteins, Sp100 And ISG20 are directly induced by interferons (IFN). PML and Sp100 proteins are covalently linked to SUMO-1, and ubiquitin-like peptide. PML NBs are disorganized in acute promyelocytic leukemia and during several DNA virus infections. In particular, the HSV-1 ICP0 protein is known to delocalize PML from NBs. Thus, NBs could play an important role in oncogenesis, IFN response and viral infections. Here, we show that HSV-1 induced PML protein degradation without altering its mRNA level. This degradation was time-and multiplicity of infectiondependent. Sp100 protein was also degraded, while another SUMO-1 conjugated protein, RanGAP1 and the IFN-induced protein kinase PKR were not. The proteasome inhibitor MG132 abrogated the HSV-1-induced PML and Sp100 degradation and partially restored their NB-localization. HSV-1 induced PML and Sp100 degradation constitutes a new example of viral inactivation of IFN target gene products.

Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-␣/␤) and type II (IFN-␥) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-␣-and IFN-␥-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.The interferon (IFN) response is one of the host response's primary defense mechanisms against viral infection. IFNs are classified as ␣, ␤, , and ␥ on the basis of their structures and antigenic properties: type I is composed of IFN-␣, -␤, and -and type II of only IFN-␥. IFN-␣/␤ is synthesized and secreted by cells in direct response to specifically viral products, including doublestranded RNA which triggers a cascade of kinase reactions and leads to the activation of specific cellular transcription factors. IFN-␣/␤ is produced by most cells as a direct response to viral infection, while IFN-␥ is synthesized almost exclusively by activated NK cells and activated T cells in response to virus-infected cells. Both type I and II IFNs achieve their antiviral effect by binding to their respective receptors (IFN-␣/␤ or IFN-␥ receptor), resulting in the activation of a distinct but related "Janus" tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway (17).Briefly, the interaction of IFN-␣/␤ with its receptors, which consist of IFNAR1 and IFNAR2 molecules, leads to the activation of the "Janus tyrosine kinases" Tyk 2 and Jak1, respectively, via tyrosine phosphorylation. Activated Tyk 2 phosphorylates IFNAR1, which then serves as a binding site for STAT2. STAT2 is then phosphorylated by Tyk 2 on tyrosine 689 and serves as a binding site for STAT1, which in turn is phosphorylated by Jak1 on tyrosine 701. The phosphorylated STATs heterodimerize; the heterodimers dissociate from the receptors and bind to the DNA-binding protein p48 (IFN regulatory factor 9 [IRF-9]) to form the complex IFN-stimulated growth factor 3 (ISGF3). The heterotrimer complexes translocate into the nucleus and bind to the IFN-stimulated response element (ISRE) to induce IFN-stimulated genes (ISGs). The binding of IFN-␥ to the IFN-␥ receptors IFNGR1 and IFNGR2 leads to the activation of the Janus kinases Jak1 and Jak2, respectively, via tyrosine phosphorylation, which in turn phosphorylate STAT1 on tyrosine. STAT1 homodimers ...

Interferons (IFNs) are a family of secreted proteins with antiviral, antiproliferative and immunomodulatory activities. The di erent biological actions of IFN are believed to be mediated by the products of speci®cally induced cellular genes in the target cells. The promyelocytic leukaemia (PML) protein localizes both in the nucleoplasm and in matrix-associated multi-protein complexes known as nuclear bodies (NBs). PML is essential for the proper formation and the integrity of the NBs. Modi®cation of PML by the Small Ubiquitin MOdi®er (SUMO) was shown to be required for its localization in NBs. The number and the intensity of PML NBs increase in response to interferon (IFN). Inactivation of the IFN-induced PML gene by its fusion to retinoic acid receptor alpha alters the normal localization of PML from the punctuate nuclear patterns of NBs to microdispersed tiny dots and results in uncontrolled growth in Acute Promyelocytic Leukaemia. The NBs-associated proteins, PML, Sp100, Sp140, Sp110, ISG20 and PA28 are induced by IFN suggesting that nuclear bodies could play a role in IFN response. Although the function of PML NBs is still unclear, some results indicate that they may represent preferential targets for viral infections and that PML could play a role in the mechanism of the antiviral action of IFNs. Viruses, which require the cellular machinery for their replication, have evolved di erent ways to counteract the action of IFN by inhibiting IFN signalling, by blocking the activities of speci®c antiviral mediators or by altering PML expression and/or localization on nuclear bodies. Oncogene (2001) 20, 7274 ± 7286.

Several pathways have been implicated in the establishment of antiviral state in response to interferon (IFN), one of which implicates the promyelocytic leukemia (PML) protein. The PML gene has been discovered 20 years ago and has led to new insights into oncogenesis, apoptosis, cell senescence, and antiviral defense. PML is induced by IFN, leading to a marked increase of expression of PML isoforms and the number of PML nuclear bodies (NBs). PML is the organizer of the NBs that contains at least 2 permanent NB-associated proteins, the IFN-stimulated gene product Speckled protein of 100 kDa (Sp100) and death-associated dead protein (Daxx), as well as numerous other transient proteins recruited in these structures in response to different stimuli. Accumulating reports have implicated PML in host antiviral defense and revealed various strategies developed by viruses to disrupt PML NBs. This review will focus on the regulation of PML and the implication of PML NBs in conferring resistance to DNA and RNA viruses. The role of PML in mediating an IFN-induced antiviral state will also be discussed.

The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed.