RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]) is the etiologic agent of Kaposi's sarcoma (KS) and lymphoproliferative diseases. We previously demonstrated that the KSHV lytic switch protein Rta stimulates DNA binding of the cellular RBP-Jk/CSL protein, the nuclear component of the Notch pathway, on Rta target promoters. In the current study, we define the promoter requirements for formation of transcriptionally productive Rta/RBP-Jk/DNA complexes. We show that highly pure Rta footprints 7 copies of a previously undescribed repetitive element in the promoter of the essential KSHV Mta gene. We have termed this element the "CANT repeat." CANT repeats are found on both strands of DNA and have a consensus sequence of ANTGTAACANT(A/T)(A/T)T. We demonstrate that Rta tetramers make high-affinity interactions (i.e., nM) with 64 bp of the Mta promoter but not single CANT units. The number of CANT repeats, their presence in palindromes, and their positions relative to the RBP-Jk binding site determine the optimal target for Rta stimulation of RBP-Jk DNA binding and formation of ternary Rta/RBP-Jk/DNA complexes. DNA binding and tetramerization mutants of Rta fail to stimulate RBP-Jk DNA binding. Our chromatin immunoprecipitation assays show that RBP-Jk DNA binding is broadly, but selectively, stimulated across the entire KSHV genome during reactivation. We propose a model in which tetramerization of Rta allows it to straddle RBP-Jk and contact repeat units on both sides of RBP-Jk. Our study integrates high-affinity Rta DNA binding with the requirement for a cellular transcription factor in Rta transactivation.

Asunto(s)

Herpesvirus Humano 8/patogenicidad , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Línea Celular , Inmunoprecipitación de Cromatina , Huella de ADN , ADN Viral/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Transactivadores/genética , Activación Viral

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are coinfected with Epstein-Barr virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA-binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen 2 (EBNA-2) to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV(+)/EBV(+) PEL cells but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA-2 growth deficiency in an autocrine/paracrine manner. Complementation of EBNA-2 deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV(+)/EBV(+) PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA-2 and Rta induce distinct alterations in the cellular proteomes that contribute to the growth of infected cells.

Asunto(s)

Herpesvirus Humano 4/fisiología , Herpesvirus Humano 8/fisiología , Receptores Notch/fisiología , Animales , Secuencia de Bases , Línea Celular , Proliferación Celular , Medios de Cultivo Condicionados , Cartilla de ADN/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Expresión Génica , Genes Virales , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/fisiología , Ratones , Ratones Noqueados , Modelos Biológicos , Regiones Promotoras Genéticas , Transducción de Señal , Transactivadores/genética , Transactivadores/fisiología , Transfección , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/fisiología , Proteínas Virales/genética , Proteínas Virales/fisiología , Activación Viral/fisiología

RESUMEN

The p53 gene product is overexpressed in approximately 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated that a modified vaccinia Ankara (MVA) vaccine expressing human p53 (MVA-p53) was moderately active when given as a homologous prime/boost in a human p53 knock in (Hupki) mouse model. We needed to improve upon the inefficient homologous boosting approach, because development of neutralizing immunity to the vaccine viral vector backbone suppresses its immunogenicity. To enhance specificity, we examined the combination of 2 different vaccine vectors provided in sequence as a heterologous prime/boost. Hupki mice were evaluated as a human p53 tolerant model to explore the capacity of heterologous p53 immunization to reject human p53-expressing tumors. We employed attenuated recombinant Listeria monocytogenes expressing human p53 (LmddA-LLO-p53) in addition to MVA-p53. Heterologous p53 immunization resulted in a significant increase in p53-specific CD8 and CD4 T cells compared with homologous single vector p53 immunization. Heterologous p53 immunization induced protection against tumor growth but had only a modest effect on established tumors. To enhance the immune response we used synthetic double-strand RNA (polyinsosinic:polycytidylic acid) and unmethylated CpG-containing oligodeoxynucleotide to activate the innate immune system via Toll-like receptors. Treatment of established tumor-bearing Hupki mice with polyinsosinic:polycytidylic acid and CpG-oligodeoxynucleotide in combination with heterologous p53 immunization resulted in enhanced tumor rejection relative to treatment with either agent alone. These results suggest that heterologous prime/boost immunization and Toll-like receptor stimulation increases the efficacy of a cancer vaccine, targeting a tolerized tumor antigen.

Asunto(s)

Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Oligodesoxirribonucleótidos/inmunología , ARN Bicatenario/inmunología , Proteína p53 Supresora de Tumor/inmunología , Adenocarcinoma/patología , Animales , Vacunas contra el Cáncer , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Vectores Genéticos/inmunología , Rechazo de Injerto , Humanos , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Receptores Toll-Like/inmunología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

PURPOSE: Transforming growth factor beta (TGFbeta) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFbeta receptor II (TGFbeta RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models. EXPERIMENTAL DESIGN: We generated neutralizing antibodies to TGFbeta RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models. RESULTS: Anti-TGFbeta RII antibody blocked TGFbeta RII binding to TGFbeta 1, 2, and 3, and attenuated the TGFbeta-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumor-bearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis. CONCLUSIONS: These preclinical data provide a foundation to support using anti-TGFbeta RII antibody as a therapeutic agent for TGFbeta RII-dependent cancer with metastatic capacity.

Asunto(s)

Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Proteína Smad2/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Listeria monocytogenes is a facultative intracellular gram-positive bacterium that naturally infects professional antigen presenting cells (APC) to target antigens to both class I and class II antigen processing pathways. This infection process results in the stimulation of strong innate and adaptive immune responses, which make it an ideal candidate for a vaccine vector to deliver heterologous antigens. This ability of L. monocytogenes has been exploited by several researchers over the past decade to specifically deliver tumor-associated antigens that are poorly immunogenic such as self-antigens. This review describes the preclinical studies that have elucidated the multiple immune responses elicited by this bacterium that direct its ability to influence tumor growth.

Asunto(s)

Vacunas contra el Cáncer/uso terapéutico , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Neoplasias/terapia , Vacunas Sintéticas/uso terapéutico , Células Presentadoras de Antígenos/inmunología , Vacunas contra el Cáncer/inmunología , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Listeriosis/microbiología , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Vacunas Sintéticas/inmunología , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunología

RESUMEN

The Kaposi's sarcoma-associated herpesvirus (KSHV) Mta protein, encoded by open reading frame 57, is a transactivator of gene expression that is essential for productive viral replication. Previous studies have suggested both transcriptional and posttranscriptional roles for Mta, but little is known regarding Mta's transcriptional function. In this study, we demonstrate that Mta cooperates with the KSHV lytic switch protein, Rta, to reactivate KSHV from latency, but Mta has little effect on reactivation when expressed alone. We demonstrate that the Mta and Rta proteins are expressed with similar but distinct kinetics during KSHV reactivation. In single-cell analyses, Mta expression coincides tightly with progression to full viral reactivation. We demonstrate with promoter reporter assays that while Rta activates transcription in all cell lines tested, Mta's ability to transactivate promoters, either alone or synergistically with Rta, is cell and promoter specific. In particular, Mta robustly transactivates the nut-1/PAN promoter independently of Rta in 293 and Akata-31 cells. Using nuclear run-on assays, we demonstrate that Mta stimulates transcriptional initiation in 293 cells. Rta and Mta physically interact in infected cell extracts, and this interaction requires the intact leucine repeat and central region of Rta in vitro. We demonstrate that Mta also binds to the nut-1/PAN promoter DNA in vitro and in infected cells. An Mta mutant with a lesion in a putative A/T hook domain is altered in DNA binding and debilitated in transactivation. We propose that one molecular mechanism of Mta-mediated transactivation is a direct effect on transcription by direct and indirect promoter association.

Asunto(s)

Herpesvirus Humano 8/fisiología , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Transcripción Genética , Proteínas Virales/metabolismo , Activación Viral , Animales , Línea Celular , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidad , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Regiones Promotoras Genéticas/genética , Transactivadores/genética , Activación Transcripcional , Proteínas Virales/genética , Latencia del Virus , Replicación Viral

RESUMEN

The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Proteínas Represoras/genética , Transactivadores/metabolismo , Activación Transcripcional , Proteínas Virales/genética , Proteínas Virales/metabolismo , Activación Viral/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Células Cultivadas , ADN/metabolismo , Fibroblastos/metabolismo , Herpesvirus Humano 8/genética , Humanos , Proteínas Inmediatas-Precoces/análisis , Proteínas Inmediatas-Precoces/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Factor 1 de Transcripción de Unión a Octámeros/análisis , Factor 1 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Elementos de Respuesta , Transactivadores/análisis , Transactivadores/genética , Transfección , Proteínas Virales/análisis

RESUMEN

The Kaposi's sarcoma-associated herpesvirus open reading frame 50 (ORF50) protein (called Rta), is necessary and sufficient for reactivation of the virus from latency. We previously demonstrated that a truncated mutant of ORF50 lacking its C-terminal transcriptional activation domain, called ORF50DeltaSTAD, formed mixed multimers with wild-type (WT) ORF50 and functioned as a dominant negative inhibitor of reactivation. For this report, we investigated the requirements for multimerization of ORF50/Rta in transactivation and viral reactivation. We analyzed multimerization of WT, mutant, and chimeric ORF50 proteins, using Blue Native polyacrylamide gel electrophoresis and size exclusion chromatography. WT and mutant ORF50 proteins form tetramers and higher-order multimers, but not monomers, in solution. The proline-rich, N-terminal leucine heptapeptide repeat (LR) of ORF50 (amino acids [aa] 244 to 275) is necessary but not sufficient for oligomer formation and functions in concert with the central portion of ORF50/Rta (aa 245 to 414). The dominant negative mutant ORF50DeltaSTAD requires the LR to form mixed multimers with WT ORF50 and inhibit its function. In the context of the WT ORF50/Rta protein, mutagenesis of the LR, or replacement of the LR by heterologous multimerization domains from the GCN4 or p53 proteins, demonstrates that tetramers of Rta are sufficient for transactivation and viral reactivation. Mutants of Rta that are unable to form tetramers but retain the ability to form higher-order multimers are reduced in function or are nonfunctional. We concluded that the proline content, but not the leucine content, of the LR is critical for determining the oligomeric state of Rta.

Asunto(s)

Herpesvirus Humano 8/química , Proteínas Inmediatas-Precoces/química , Estructura Cuaternaria de Proteína , Sarcoma de Kaposi/virología , Transactivadores/química , Proteínas Virales/química , Activación Viral , Secuencia de Aminoácidos , Línea Celular , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/fisiología , Leucina/química , Leucina/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta/genética , Prolina/química , Prolina/genética , Transactivadores/genética , Transactivadores/fisiología , Proteínas Virales/genética , Proteínas Virales/fisiología , Activación Viral/genética

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.

Asunto(s)

ADN Viral/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Lectinas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Proteínas Virales/metabolismo , Empalme Alternativo/genética , Secuencia de Bases , Línea Celular , ADN Viral/genética , Regulación Viral de la Expresión Génica , Genoma Viral/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Transactivadores/genética , Transcripción Genética/genética , Proteínas Virales/genética