RESUMEN
HIV-1 integrase (HIV-1 IN), a key element of HIV-1-derived lentiviral vectors, is crucial for the stable maintenance of the vector gene by inserting them into host genome. HIV-1 IN has been found to have functions other than integration, such as involving in virion morphology, viral DNA synthesis and viral DNA nuclear import. In our study, the yeast two-hybrid assay identified a tetrapeptide 156KELK159 in HIV-1 IN that was crucial for HIV-1 IN and Daxx interaction. To investigate the functions of the tetrapeptide 156KELK159 of the HIV-1 IN, both the wild type HIV-1 IN and a mutant without 156KELK159 were used to package the EGFP reporter gene contained lentivirus. p24 based titer assay revealed that deleting the tetrapeptide did not affect virus packaging. The result was verified by quantitative real time PCR with viral specific primers. But the 156KELK159 was crucial for lentiviral gene integration. Deleting the tetrapeptide made the percentage of cells expressing the reporter gene significantly decreased and did not affect the level of DNA entered into the cells or nucleus. Real time reverse transcription PCR and FACS were used to detect the lentiviral report gene expression in infection maintaining cells and revealed 156KELK159 did not affect lentiviral vector gene expression. Our results may shed light on the regulatory mechanism of gene integration of lentivirus.
Asunto(s)
Integrasa de VIH/genética , VIH-1/genética , Oligopéptidos/fisiología , Transducción Genética/métodos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Proteínas Co-Represoras , Cartilla de ADN/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Oligopéptidos/genética , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
Phage PhiC31 integrase-mediated gene delivery is believed to be safer than using retroviral vectors since the protein confines its insertion of the target gene to a limited number of sites in mammalian genomes. To evaluate its safety in human cells, it is important to understand the interactions between this integrase and cellular proteins. Here we show that PhiC31 integrase interacts with TTRAP as presented by yeast two-hybrid and co-immunoprecipitation assays. Reducing the expression of endogenous TTRAP can increase the efficiency of PhiC31 integrase-mediated integration. A possible effect of interaction between PhiC31 integrase and TTRAP was highlighted by the fact that PhiC31 integrase inhibited the NFkappaB activation mediated by IL-1 in a dose-dependent manner. Because low dose of PhiC31 integrase can mediate considerable recombination events, we suggest that low dose of PhiC31 integrase be used when this integrase is applied in human cells.
Asunto(s)
Bacteriófagos/enzimología , Integrasas/metabolismo , FN-kappa B/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Bacteriófagos/efectos de los fármacos , Células HeLa , Humanos , Interleucina-1/farmacología , Unión Proteica/efectos de los fármacos , Recombinación Genética/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Integración Viral/efectos de los fármacosRESUMEN
PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-gamma, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Línea Celular , Proteínas Co-Represoras , Proteínas de Unión al ADN , Humanos , Interferón gamma/farmacología , Chaperonas Moleculares , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas , Proteína de la Leucemia Promielocítica , Factores de Transcripción/genética , Transcripción Genética , LevadurasRESUMEN
The death-associated protein Daxx is a ubiquitously expressed gene in mammals and is widely involved in transcriptional regulation and cellular intrinsic immune response against incoming virus. We found here that knocking down endogenous Daxx with specific siRNA increased HIV-1-derived lentiviral reporter gene expression in 293T cells. This repressive effect of Daxx is not due to its inhibition on viral gene integration into the cellular genome and is independent of the ubiquitin promoter on the vFUGW lentiviral vector. Instead, this inhibition is dependent on Daxx's interaction with HIV-1 integrase. A histone deacetylases (HDACs) inhibitor increased reporter gene expression to the level similar to Daxx knockdown in vFUGW infected cells but there was no additive effect in combination of HDACs inhibitor and Daxx-specific siRNA. Our results suggest that Daxx may associate with HIV-1-derived lentiviral DNA via interacting with HIV-1 integrase and recruit HDACs to viral DNA to repress lentiviral gene expression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Viral/metabolismo , Regulación Viral de la Expresión Génica , Integrasa de VIH/metabolismo , VIH-1/genética , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Proteínas Co-Represoras , Genes Reporteros , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina/genéticaRESUMEN
Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.
