ABSTRACT
Kaposi sarcoma-associated herpesvirus is associated with two lymphoproliferative disorders, primary effusion lymphoma (PEL) and Castleman disease. In PEL, Kaposi sarcoma-associated herpesvirus is present in a latent form expressing only few viral genes. Among them is a viral homologue of cellular interferon regulatory factors, vIRF-3. To study the role of vIRF-3 in PEL lymphomagenesis, we analyzed the interaction of vIRF-3 with cellular proteins. Using yeast two-hybrid screen, we detected the association between vIRF-3 and c-Myc suppressor, MM-1alpha. The vIRF-3 and MM-1alpha interaction was also demonstrated by glutathione S-transferase pulldown assay and coimmunoprecipitation of endogenous vIRF-3 and MM-1alpha in PEL-derived cell lines. Overexpression of vIRF-3 enhanced the c-Myc-dependent transcription of the gene cdk4. Addressing the molecular mechanism of the vIRF-3-mediated stimulation, we demonstrated that the association between MM-1alpha and c-Myc was inhibited by vIRF-3. Furthermore, the recruitment of vIRF-3 to the cdk4 promoter and the elevated levels of the histone H3 acetylation suggest the direct involvement of vIRF-3 in the activation of c-Myc-mediated transcription. These findings indicate that vIRF-3 can effectively stimulate c-Myc function in PEL cells and consequently contribute to de-regulation of B-cell growth and differentiation.
Subject(s)
Herpesvirus 8, Human/metabolism , Interferon Regulatory Factor-3/metabolism , Interferon Regulatory Factors/metabolism , Lymphoma, Primary Effusion/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Viral Proteins/metabolism , Cell Line, Tumor , Gene Library , Humans , Two-Hybrid System TechniquesABSTRACT
Kaposi's sarcoma-associated herpesvirus has been linked to Kaposi's sarcoma, body cavity-based lymphoma, and Castleman's disease. The Kaposi's sarcoma-associated herpesvirus genome contains a cluster of open reading frames encoding proteins (vIRFs) with homology to the cellular transcription factors of the interferon regulatory factor family. vIRF-3, also called LANA2, is a latently expressed nuclear protein. Here we demonstrate that vIRF-3 directly interacts with cellular interferon regulatory factor (IRF) IRF-3, IRF-7, and the transcriptional co-activator CBP/p300. The mapping of the vIRF-3 binding domain revealed that vIRF-3 associates with both IRF-3 and IRF-7 through its C-terminal region. The p300 domain, which interacts with vIRF-3, is distinct from the previously identified IBiD domain, to which both vIRF-1 and IRF-3 bind. Thus, in contrast to vIRF-1, vIRF-3 neither blocks the interaction between IRF-3 and p300 nor inhibits the histone acetylation. Although vIRF-3 is not a DNA-binding protein, it is recruited to the IFNA promoters via its interaction with IRF-3 and IRF-7. The presence of vIRF-3 in the enhanceosome assembled on the IFNA promoters increases binding of IRF-3, IRF-7, and acetylated histone H3 to this promoter region. Consequently, vIRF-3 stimulates the IRF-3- and IRF-7-mediated activation of type I interferon (IFNA and IFNB) genes and the synthesis of biologically active type I interferons in infected B cells. These studies illustrate that vIRF-3 and vIRF-1 have clearly distinct functions. In addition to its co-repressor activity, vIRF-3 can also act as a transcriptional activator on genes controlled by cellular IRF-3 and IRF-7.
Subject(s)
DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Herpesvirus 8, Human/genetics , Transcription Factors/metabolism , Transcription Factors/pharmacology , Transcription, Genetic/drug effects , Acetylation , Binding Sites , Cell Line , DNA/metabolism , DNA-Binding Proteins/genetics , Drug Interactions , Gene Expression , HeLa Cells , Histones/metabolism , Humans , Immunosorbent Techniques , Interferon Regulatory Factor-3 , Interferon Regulatory Factor-7 , Interferon Regulatory Factors , Interferon Type I/biosynthesis , Interferon Type I/genetics , Lymphoma , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Trans-Activators/metabolism , Transcription Factors/genetics , Transfection , Tumor Cells, Cultured , Viral ProteinsABSTRACT
We have previously shown a critical role for IFN regulatory factor 5 (IRF-5) in the innate immune response to virus infection. For the first time, we now show that although IRF-5 is a direct target of p53, its cell cycle regulatory and proapoptotic effects are p53 independent. IRF-5 inhibits both in vitro and in vivo B-cell lymphoma tumor growth in the absence of wild-type p53. The molecular mechanism(s) of IRF-5-mediated growth inhibition is associated with a G(2)-M cell cycle arrest and modulation of growth regulatory and proapoptotic genes, including p21, Bak, DAP kinase 2, and Bax. Taken together, these data indicate that although IRF-5 is a downstream target of p53, its growth inhibitory and proapoptotic effects are independent of p53.
Subject(s)
Apoptosis/physiology , Cell Cycle/physiology , DNA-Binding Proteins/physiology , Proto-Oncogene Proteins c-bcl-2 , Transcription Factors/physiology , Animals , Caspase 8 , Caspase 9 , Caspases/biosynthesis , Caspases/genetics , Cell Division/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , Cyclins/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Female , G2 Phase/physiology , HCT116 Cells , Humans , Interferon Regulatory Factor-7 , Interferon Regulatory Factors , Leukemia/genetics , Leukemia/metabolism , Leukemia/pathology , Leukocytes, Mononuclear/metabolism , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/metabolism , Lymphoma, B-Cell/pathology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Nude , Mitosis/physiology , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Signal Transduction/physiology , Transcription Factors/biosynthesis , Transcription Factors/genetics , Tumor Suppressor Protein p53/physiology , bcl-2 Homologous Antagonist-Killer Protein , bcl-2-Associated X ProteinABSTRACT
Transcription factors of the interferon regulatory factor (IRF) family have been identified as critical mediators of early inflammatory gene transcription in infected cells. We recently determined that, besides IRF-3 and IRF-7, IRF-5 serves as a direct transducer of virus-mediated signaling. In contrast to that mediated by the other two IRFs, IRF-5-mediated activation is virus specific. We show that, in addition to Newcastle disease virus (NDV) infection, vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) infection activates IRF-5, leading to the induction of IFNA gene subtypes that are distinct from subtypes induced by NDV. The IRF-5-mediated stimulation of inflammatory genes is not limited to IFNA since in BJAB/IRF-5-expressing cells IRF-5 stimulates transcription of RANTES, macrophage inflammatory protein 1 beta, monocyte chemotactic protein 1, interleukin-8, and I-309 genes in a virus-specific manner. By transient- transfection assay, we identified constitutive-activation (amino acids [aa] 410 to 489) and autoinhibitory (aa 490 to 539) domains in the IRF-5 polypeptide. We identified functional nuclear localization signals (NLS) in the amino and carboxyl termini of IRF-5 and showed that both of these NLS are sufficient for nuclear translocation and retention in infected cells. Furthermore, we demonstrated that serine residues 477 and 480 play critical roles in the response to NDV infection. Mutation of these residues from serine to alanine dramatically decreased phosphorylation and resulted in a substantial loss of IRF-5 transactivation in infected cells. Thus, this study defines the regulatory phosphorylation sites that control the activity of IRF-5 in NDV-infected cells and provides further insight into the structure and function of IRF-5. It also shows that the range of IRF-5 immunoregulatory target genes includes members of the cytokine and chemokine superfamilies.
