RESUMEN
Type-I interferons (IFN-I) are the innate immune system's principal defense against viral infections. Human immunodeficiency virus-1 (HIV-1) has evolved several ways to suppress or evade the host's innate immunity in order to survive and replicate to sustain infection. Suppression of IFN-I is one among the multiple escape strategies used by HIV-1 to prevent its clearance. HIV-1 protease which helps in viral maturation has also been observed to cleave host cellular protein kinases. In this study we performed a comprehensive screening of a human kinase library using AlphaScreen assay and identified that TANK binding kinase-1 (TBK1) was cleaved by HIV-1 protease (PR). We demonstrate that PR cleaved TBK1 fails to phosphorylate IFN regulatory factor 3 (IRF3), thereby reducing the IFN-I promoter activity and further reveal that the PR mediated suppression of IFN-I could be counteracted by protease inhibitors (PI) in vitro. We have also revealed that mutations of HIV-1 PR that confer drug resistance to PIs reduce the enzyme's ability to cleave TBK1. The findings of this study unearth a direct link between HIV-1 PR activity and evasion of innate immunity by the virus, the possible physiological relevance of which warrants to be determined.
RESUMEN
Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Línea Celular Tumoral , Células HEK293 , VIH-1/patogenicidad , Humanos , Mutación , Unión Proteica , Proteómica , ARN Interferente Pequeño/metabolismo , Ensamble de Virus/fisiologíaRESUMEN
Virus-host interactions play vital roles in viral replication and virus-induced pathogenesis. Viruses rely entirely upon host cells to reproduce progeny viruses; however, host factors positively or negatively regulate virus replication by interacting with viral proteins. The elucidation of virus-host protein interaction not only provides a better understanding of the molecular mechanisms by which host cells combat viral infections, but also facilitates the development of new anti-viral therapeutics. Identification of relevant host factors requires techniques that enable comprehensive characterization of virus-host protein interactions. In this study, we developed a proteomic approach to systematically identify human protein kinases that interact potently with viral proteins. For this purpose, we synthesized 412 full-length human protein kinases using the wheat germ cell-free protein synthesis system, and screened them for their association with a virus protein using the amplified luminescent proximity homogenous assay (AlphaScreen). Using this system, we attempted to discover a robust anti-viral host restriction mechanism targeting virus protein X (Vpx) of HIV-2. The screen identified H11/HSPB8 as a Vpx-binding protein that negatively regulates the stability and function of Vpx. Indeed, overexpression of H11/HSPB8 promoted the degradation of Vpx via the ubiquitin-proteasome pathway and inhibited its interaction with SAMHD1, a host restriction factor responsible for blocking replication of HIV. Conversely, targeted knockdown of H11/HSPB8 in human trophoblast cells, which ordinarily express high levels of this protein, restored the expression and function of Vpx, making the cells highly susceptible to viral replication. These results demonstrate that our proteomic approach represents a powerful tool for revealing virus-host interaction not yet identified by conventional methods. Furthermore, we showed that H11/HSPB8 could be a potential host regulatory factor that may prevent placental infection of HIV-2 during pregnancy.
RESUMEN
APOBEC3G (A3G) is an innate antiviral restriction factor that strongly inhibits the replication of human immunodeficiency virus type 1 (HIV-1). An HIV-1 accessory protein, Vif, hijacks the host ubiquitin-proteasome system to execute A3G degradation. Identification of the host pathways that obstruct the action of Vif could provide a new strategy for blocking viral replication. We demonstrate here that the host protein ASK1 (apoptosis signal-regulating kinase 1) interferes with the counteraction by Vif and revitalizes A3G-mediated viral restriction. ASK1 binds the BC-box of Vif, thereby disrupting the assembly of the Vif-ubiquitin ligase complex. Consequently, ASK1 stabilizes A3G and promotes its incorporation into viral particles, ultimately reducing viral infectivity. Furthermore, treatment with the antiretroviral drug AZT (zidovudine) induces ASK1 expression and restores the antiviral activity of A3G in HIV-1-infected cells. This study thus demonstrates a distinct function of ASK1 in restoring the host antiviral system that can be enhanced by AZT treatment.
Asunto(s)
Citidina Desaminasa/metabolismo , VIH-1/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Virión/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Fármacos Anti-VIH/farmacología , Citidina Desaminasa/efectos de los fármacos , Células HEK293 , VIH-1/efectos de los fármacos , Humanos , Inmunoprecipitación , MAP Quinasa Quinasa Quinasa 5/efectos de los fármacos , Ubiquitinación , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Zidovudina/farmacologíaRESUMEN
Cancer stem cells (CSCs) retain the capacity to propagate themselves through self-renewal and to produce heterogeneous lineages of cancer cells constituting the tumor. Novel drugs that target CSCs can potentially eliminate the tumor initiating cell population therefore resulting in complete cure of the cancer. We recently established a CSC-like model using induced pluripotent stem cell (iPSC) technology to reprogram and partially differentiate human mammary epithelial MCF-10A cells. Using the induced CSC-like (iCSCL) model, we developed a phenotypic drug assay system to identify agents that inhibit the stemness and self-renewal properties of CSCs. The selectivity of the agents was assessed using three distinct assays characterized by cell viability, cellular stemness and tumor sphere formation. Using this approach, we found that withaferin A (WA), an Ayurvedic medicine constituent, was a potent inhibitor of CSC stemness leading to cellular senescence primarily via the induction of p21Cip1 expression. Moreover, WA exhibited strong anti-tumorigenic activity against the iCSCL. These results indicate that our iCSCL model provides an innovative high throughput platform for a simple, easy, and cost-effective method to search for novel CSC-targeting drugs. Furthermore, our current study identified WA as a putative drug candidate for abrogating the stemness and tumor initiating ability of CSCs.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Witanólidos/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Reprogramación Celular , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Factores de Tiempo , TransfecciónRESUMEN
Human parainfluenza virus 3 (HPIV3) commonly causes respiratory disorders in infants and young children. Monoclonal antibodies (MAbs) have been produced to several components of HPIV3 and commercially available. However, the utility of these antibodies for several immunological and proteomic assays for understanding the nature of HPIV3 infection remain to be characterized. Herein, we report the development and characterization of MAbs against hemagglutinin-neuraminidase (HN) of HPIV3. A recombinant full-length HPIV3-HN was successfully synthesized using the wheat-germ cell-free protein production system. After immunization and cell fusion, 36 mouse hybridomas producing MAbs to HPIV3-HN were established. The MAbs obtained were fully characterized using ELISA, immunoblotting, and immunofluorescent analyses. Of the MAbs tested, single clone was found to be applicable in both flow cytometry and immunoprecipitation procedures. By utilizing the antibody, we identified HPIV3-HN binding host proteins via immunoprecipitation-based mass spectrometry analysis. The newly-developed MAbs could thus be a valuable tool for the study of HPIV3 infection as well as the several diagnostic tests of this virus.
RESUMEN
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication. RESULTS: A proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages. CONCLUSION: Our current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.
Asunto(s)
VIH-1/fisiología , Interacciones Huésped-Patógeno , Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Fosforilación , Virión/metabolismoRESUMEN
Expression of Epstein-Barr Virus BZLF1, a key protein initiating the switch from latent to lytic infection, is known to cause cell growth arrest by accumulating p53 and p21(WAF1/CIP1) in epithelial cells, but its molecular mechanism remains elusive. We found here that the BZLF1 protein stimulates p53 binding to its recognition sequence. The BZLF1 accelerated the rate of p53-DNA complex formation through the interaction with p53 protein and also enhanced p53-specific transcription in vitro. Furthermore, p53 protein was found to bind to its target promoter regions specifically in the early stages of lytic replication. Overexpression of p53 at the early stages of lytic replication enhanced viral genome replication, supporting the idea that p53 plays an important role in the initiation steps of EBV replication. Taking the independent role of BZLF1 on p53 degradation into consideration, we propose that the BZLF1 protein regulates p53 and its target gene products in two distinctive manners; transient induction of p53 at the early stages for the initiation of viral productive replication and p53 degradation at the later stages for S-phase like environment preferable for viral replication.
Asunto(s)
Herpesvirus Humano 4/genética , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Replicación Viral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Viral de la Expresión Génica , Células HeLa , Humanos , Activación TranscripcionalRESUMEN
p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (Elongin B/C-Cul2/5-SOCS-box protein) ubiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response by viral lytic replication enhances its binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could be shown to catalyze ubiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhibits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated by activated DNA damage signaling during viral lytic infection.
Asunto(s)
Herpesvirus Humano 4/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Herpesvirus Humano 4/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , Fosforilación , Ubiquitinación , Replicación ViralRESUMEN
The Epstein-Barr virus (EBV) BMRF1 protein is an essential replication protein acting at viral replication forks as a viral DNA polymerase processivity factor, whereas the BALF2 protein is a single-stranded DNA-binding protein that also acts at replication forks and is most abundantly expressed during viral productive replication. Here we document that the BMRF1 protein evidently enhances viral BZLF1 transcription factor-mediated transactivation of the BALF2 gene promoter. Mutagenesis and electrophoretic mobility shift assays demonstrated the BALF2 promoter to harbor two BZLF1 protein-binding sites (BZLF1-responsive elements). Direct binding of the BZLF1 protein to BZLF1-responsive elements and physical interaction between BZLF1 and BMRF1 proteins are prerequisite for the BMRF1 protein up-regulation of the BALF2 gene promoter. A monomeric mutant, C95E, which is defective in homodimerization, could still interact and enhance BZLF1-mediated transactivation. Furthermore although EBV protein kinase phosphorylates BMRF1 protein extensively, it turned out that phosphorylation of the protein by the kinase is inhibitory to the enhancement of the BZLF1-mediated transactivation of BALF2 promoter. Exogenous expression of BMRF1 protein augmented BALF2 expression in HEK293 cells harboring the EBV genome but lacking BMRF1 and BALF5 genes, demonstrating functions as a transcriptional regulator in the context of viral infection. Overall the BMRF1 protein is a multifunctional protein that cannot only act as a DNA polymerase processivity factor but also enhances BALF2 promoter transcription as a coactivator for the BZLF1 protein, regulating the expression level of viral single-stranded DNA-binding protein.
Asunto(s)
Antígenos Virales/metabolismo , Proteínas de Unión al ADN/biosíntesis , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Proteínas Virales/biosíntesis , Secuencia de Bases , Unión Competitiva , Línea Celular Tumoral , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Humanos , Luciferasas/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Transactivadores/metabolismo , Proteínas Virales/genéticaRESUMEN
Epstein-Barr virus (EBV) productive replication occurs in an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. The EBV protein kinase (PK), encoded by the viral BGLF4 gene, is a Ser/Thr protein kinase, which phosphorylates both viral and cellular proteins, modifying the cellular environment for efficient viral productive replication. We here provide evidence that the EBV PK phosphorylates the CDK inhibitor p27(Kip1), resulting in ubiquitination and degradation in a proteasome-dependent manner during EBV productive replication. Experiments with BGLF4 knockdown by small interfering RNA and BGLF4 knock-out viruses clarified that EBV PK is involved in p27(Kip1) degradation upon lytic replication. Transfection of the BGLF4 expression vector revealed that EBV PK alone could phosphorylate the Thr-187 residue of p27(Kip1) and that the ubiquitination and degradation of p27(Kip1) occurred in an SCF(Skp2) ubiquitin ligase-dependent manner. In vitro, EBV PK proved capable of phosphorylating p27(Kip1) at Thr-187. Unlike cyclin E-CDK2 activity, the EBV PK activity was not inhibited by p27(Kip1). Overall, EBV PK enhances p27(Kip1) degradation effectively upon EBV productive replication, contributing to establishment of an S-phase-like cellular environment with high CDK activity.
Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Vectores Genéticos , Células HeLa , Humanos , Fosforilación , Plásmidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/metabolismo , Fase S , Treonina/química , Ubiquitina/química , Proteínas Virales/metabolismoRESUMEN
The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.
Asunto(s)
Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Línea Celular , Replicación del ADN , ADN Viral/biosíntesis , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Genoma Viral , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiología , Humanos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Virales/genéticaRESUMEN
Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.
Asunto(s)
Reparación del ADN , Herpesvirus Humano 4/fisiología , Recombinasa Rad51/metabolismo , Proteína de Replicación A/metabolismo , Replicación Viral , Animales , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Replicación del ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Genoma Viral , Herpesvirus Humano 4/genética , Humanos , Proteína Homóloga de MRE11 , Fosforilación , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Recombinación GenéticaRESUMEN
The Epstein-Barr virus (EBV) lytic program elicits ATM-dependent DNA damage response, resulting in phosphorylation of p53 at N-terminus, which prevents interaction with MDM2. Nevertheless, p53-downstream signaling is blocked. We found here that during the lytic infection p53 was actively degraded in a proteasome-dependent manner even with a reduced level of MDM2. BZLF1 protein enhanced the ubiquitination of p53 in SaOS-2 cells. The degradation of p53 was observed even in the presence of Nutlin-3, an inhibitor of p53-MDM2 interaction, and also in mouse embryo fibroblasts lacking mdm2 gene, indicating that the BZLF1 protein-induced degradation of p53 was independent of MDM2. Furthermore, Nutlin-3 increased the level of p53 in the latent phase of EBV infection but not in the lytic phase. Although p53 level is regulated by MDM2 in the latent phase, it might be mediated by the BZLF1 protein-associated E3 ubiquitin ligase in the lytic phase for efficient viral propagation.
Asunto(s)
Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 4/fisiología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Regulación hacia Abajo , Humanos , Leupeptinas , Ratones , Transactivadores/genética , Transcripción GenéticaRESUMEN
Reactivation of the Epstein-Barr virus from latency is dependent on expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical inducers such as 12-O-tetradecanoylphorbol-13-acetate and calcium ionophore. We found here that Transducer of Regulated cAMP-response Element-binding Protein (CREB) (TORC) 2 enhances Zp activity 10-fold and more than 100-fold with co-expression of the BZLF1 protein. Mutational analysis of Zp revealed that the activation by TORC is dependent on ZII and ZIII cis elements, binding sites for CREB family transcriptional factors and the BZLF1 protein, respectively. Immunoprecipitation, chromatin immunoprecipitation, and reporter assay using Gal4-luc and Gal4BD-BZLF1 fusion protein indicate that TORC2 interacts with BZLF1, and that the complex is efficiently recruited onto Zp. These observations clearly indicate that TORC2 activates the promoter through interaction with the BZLF1 protein as well as CREB family transcriptional factors. Induction of the lytic replication resulted in the translocation of TORC2 from cytoplasm to viral replication compartments in nuclei, and furthermore, activation of Zp by TORC2 was augmented by calcium-regulated phosphatase, calcineurin. Silencing of endogenous TORC2 gene expression by RNA interference decreased the levels of the BZLF1 protein in response to 12-O-tetradecanoylphorbol-13-acetate/ionophore. Based on these results, we conclude that Epstein-Barr virus exploits the calcineurin-TORC signaling pathway through interactions between TORC and the BZLF1 protein in reactivation from latency.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Herpesvirus Humano 4/fisiología , Elementos de Respuesta/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología , Carcinógenos/farmacología , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación Viral de la Expresión Génica/efectos de los fármacos , Regulación Viral de la Expresión Génica/fisiología , Humanos , Mutación , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Acetato de Tetradecanoilforbol/farmacología , Transactivadores/genética , Factores de Transcripción/genética , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacosRESUMEN
DNA damage induces hyper-phosphorylation of the Sp1 transcriptional factor. We have demonstrated that ionizing radiation-associated DNA double-strand breaks (DSBs) induce phosphorylation of at least Ser-56 and Ser-101 residues on Sp1 in an ATM-dependent manner. UV irradiation- or hydroxyurea (HU)-induced replicative stress results in phosphorylation of only the Ser-101 residue. Furthermore, silencing of the ATM- and Rad3-related protein (ATR) in ATM-deficient cells treated with HU abrogated the Ser-101 phosphorylation. Thus, phosphorylation of Ser-101 on Sp1 appears to be a general response to DNA damage dependent on both ATM and ATR. Although silencing of Sp1 expression by siRNA targeting resulted in an increase in sensitivity to ionizing radiation (IR), the Ser-101 phosphorylation did not affect transcriptional activity from the Sp1 responsive promoter. Confocal laser microscopy analysis revealed co-localization of phosphorylated Sp1 at Ser-101 with phosphorylated ATM at Ser-1981, the affected sites representing DSBs. These observations suggest that phosphorylated Sp1 might play a role in DNA repair at damage sites rather than functioning in transcriptional regulation.
Asunto(s)
Daño del ADN , Factor de Transcripción Sp1/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Núcleo Celular/enzimología , Núcleo Celular/efectos de la radiación , Roturas del ADN de Doble Cadena/efectos de la radiación , Replicación del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Drosophila , Activación Enzimática/efectos de la radiación , Humanos , Fosforilación/efectos de la radiación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Radiación Ionizante , Factor de Transcripción Sp1/genética , Transcripción Genética/efectos de la radiación , Proteínas Supresoras de Tumor/metabolismoRESUMEN
PURPOSE: In the treatment of head and neck malignancy, cisplatin and 5-FU have been used the most as chemotherapeutic agents. The difference in efficacies of these is unclear and controversial. To investigate more effective schedule, we analyzed the cytotoxicity in different treatment sequence with two agents in vitro and the mechanism for different effectiveness. METHODS: UM-SCC-23 and UM-SCC-81B, head and neck squamous cell carcinoma cell lines, were analyzed for cellular killing in alternative sequence treatment with cisplatin and 5-FU. The treatment schedule was designed based on the clinical regimen. To determine the mechanism for the difference of cytotoxicity with each schedule, cell cycle distributions of both cells after 5-FU treatment with various durations were analyzed by flow-cytometry and immunostaining with anti-PCNA and anti-BrdU. RESULTS: 5-FU pretreatment followed by cisplatin treatment showed higher cell killing in both types of cells than the reverse treatment schedule. In the cell cycle analysis and immunostaining after the treatment of 5-FU, the rate of PCNA-positive cells was increased from 24 to 144 h in both cells. The rate of BrdU-positive cells of UM-SCC-81B in flow-cytometry was also increased, while that of UM-SCC-23 was gradually decreased. These data suggested that the cells treated with 5-FU for more than 144 h were still in the S-phase with or without DNA synthesis. CONCLUSIONS: In head and neck carcinoma cells, we showed 5-FU pretreatment enhanced cisplatin cytotoxicity. The result of cell cycle analysis and immunostaining showed S-phase arrest by treatment of prolonged 5-FU treatment. The very long arrest in S-phase might be a mechanism to enhance cisplatin cytotoxicity by 5-FU pretreatment. We thus suggest pretreatment with 5-FU to enhance the effectiveness of cisplatin-based chemotherapy.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Cisplatino/farmacología , Fluorouracilo/farmacología , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Células Escamosas/tratamiento farmacológico , Bromodesoxiuridina/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Neoplasias de Células Escamosas/patología , Antígeno Nuclear de Célula en Proliferación/análisis , Antígeno Nuclear de Célula en Proliferación/metabolismoRESUMEN
During productive infection, human cytomegalovirus (HCMV) UL44 transcription initiates at three distinct start sites that are differentially regulated. Two of the start sites, the distal and the proximal, are active at early times, whereas the middle start site is active only at late times after infection. The UL44 early viral gene product is essential for viral DNA synthesis. The UL44 gene product from the late viral promoter affects primarily viral gene expression at late times after infection rather than viral DNA synthesis (H. Isomura, M. F. Stinski, A. Kudoh, S. Nakayama, S. Iwahori, Y. Sato, and T. Tsurumi, J. Virol. 81:6197, 2007). The UL44 early viral promoters have a canonical TATA sequence, "TATAA." In contrast, the UL44 late viral promoter has a noncanonical TATA sequence. Using recombinant viruses, we found that the noncanonical TATA sequence is required for the accumulation of late viral transcripts. The GC boxes that surround the middle TATA element did not affect the kinetics or the start site of UL44 late transcription. Replacement of the distal TATA element with a noncanonical TATA sequence did not affect the kinetics of transcription or the transcription start site, but it did induce an alternative transcript at late times after infection. The data indicate that a noncanonical TATA box is used at late times after HCMV infection.
Asunto(s)
Citomegalovirus/genética , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica , TATA Box/fisiología , Transcripción Genética , Proteínas Virales/genética , Secuencia de Bases , Células Cultivadas , Citomegalovirus/enzimología , Humanos , Cinética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , ARN Viral/metabolismo , TATA Box/genética , Sitio de Iniciación de la TranscripciónRESUMEN
The promoter of the major immediate-early (MIE) genes of human cytomegalovirus (HCMV), also referred to as the CMV promoter, possesses a cis-acting element positioned downstream of the TATA box between positions -14 and -1 relative to the transcription start site (+1). We determined the role of the cis-acting element in viral replication by comparing recombinant viruses with the cis-acting element replaced with other sequences. Recombinant virus with the simian CMV counterpart replicated efficiently in human foreskin fibroblasts, as well as wild-type virus. In contrast, replacement with the murine CMV counterpart caused inefficient MIE gene transcription, RNA splicing, MIE and early viral gene expression, and viral DNA replication. To determine which nucleotides in the cis-acting element are required for efficient MIE gene transcription and splicing, we constructed mutations within the cis-acting element in the context of a recombinant virus. While mutations in the cis-acting element have only a minor effect on in vitro transcription, the effects on viral replication are major. The nucleotides at -10 and -9 in the cis-acting element relative to the transcription start site (+1) affect efficient MIE gene transcription and splicing at early times after infection. The cis-acting element also acts as a cis-repression sequence when the viral IE86 protein accumulates in the infected cell. We demonstrate that the cis-acting element has an essential role in viral replication.