RESUMEN

Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4(+) T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4(+) T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4(+) T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4(+) T lymphocytes harboring a latent provirus.

Asunto(s)

Linfocitos T CD4-Positivos/efectos de los fármacos , Ciclinas/biosíntesis , Interleucina-2/farmacología , Interleucina-6/farmacología , Proteínas Serina-Treonina Quinasas/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Ciclina T , Quinasa 9 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN , Humanos , Inmunofenotipificación , Activación de Linfocitos , Factor B de Elongación Transcripcional Positiva

RESUMEN

Cdk9 is the catalytic subunit of TAK (cyclinT1/P-TEFb), a cellular protein kinase that mediates human immunodeficiency virus type 1 (HIV-1) Tat transcriptional activation function. To examine Cdk9 function in cells relevant to HIV-1 infection, we used a murine leukemia virus retrovirus vector to transduce and overexpress the cDNA of a dominant negative mutant Cdk9 protein (Cdk9-dn) in Jurkat T cells and U937 promonocytic cells. In Jurkat cells, overexpression of Cdk9-dn specifically inhibited Tat transactivation and HIV-1 replication but had no inhibitory effect on induction of CD69, CD25, and interleukin-2 following T-cell activation. In U937 cells, overexpression of Cdk9-dn sensitized cells to apoptosis, especially after phorbol myristate acetate (PMA) treatment to induce differentiation to macrophage-like cells. Because Cdk9 function is induced in PMA-treated U937 cells, Cdk9 may play an antiapoptotic role during monocyte differentiation.

Asunto(s)

Apoptosis , Quinasas Ciclina-Dependientes/fisiología , Monocitos/fisiología , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos CD4/análisis , Quinasa 9 Dependiente de la Ciclina , Productos del Gen tat/fisiología , Duplicado del Terminal Largo de VIH , Humanos , Interleucina-2/biosíntesis , Células Jurkat , Lectinas Tipo C , Receptores de Interleucina-2/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Activación Transcripcional , Células U937

RESUMEN

Cyclin T1 (CycT1) is a regulatory subunit of a general RNA polymerase II (RNAP II) elongation factor termed P-TEFb. The human immunodeficiency virus Tat protein directly associates with CycT1 to utilize CycT1/P-TEFb (also called TAK) for activation of RNAP II elongation of the integrated proviral genome. CycT1 mRNA and protein levels are induced in activated human peripheral blood lymphocytes and CycT1 protein levels are induced by a post-transcriptional mechanism when human U937 promonocytic cells are stimulated to differentiate into macrophage-like cells. To investigate mechanisms that regulate CycT1 RNA expression, we isolated the CycT1 promoter. Multiple transcription start sites were identified within 330 nucleotides upstream of the ATG initiation codon at +1. The CycT1 promoter lacks a TATA element and possesses high constitutive activity in plasmid transfection assays. Two distinct regions of the promoter were identified upstream of +1 that contain critical regulatory elements for CycT1 promoter function.

Asunto(s)

Ciclinas/genética , ADN/aislamiento & purificación , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Sitios de Unión , Ciclina T , ADN/química , ADN/metabolismo , Células HL-60 , Células HeLa , Humanos , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Transcripción Genética

RESUMEN

CDK9 is the catalytic subunit of a general RNA polymerase II (RNAP II) elongation factor termed p-TEFb which is targeted by the human immunodeficiency virus (HIV) Tat protein to activate elongation of the integrated proviral genome. CDK9 mRNA and protein levels have been observed to be induced in activated peripheral blood lymphocytes, a cell type relevant to HIV infection. To investigate mechanisms that regulate CDK9 RNA expression, we isolated genomic sequences containing the human CDK9 gene and found that CDK9 coding sequences are interrupted by six introns. There is a major transcriptional start site located 79 nucleotides upstream of the ATG initiator codon at nucleotide +1. Nucleotides -352 to -1 contain all the transcriptional regulatory elements needed for full promoter activity in transient expression assays. The CDK9 promoter contains features characteristic of a housekeeping gene, including GC-rich sequences and absence of a functional TATA element. The CDK9 promoter possesses high constitutive activity and may therefore have utility in expression vectors or gene therapy vectors.

Asunto(s)

Quinasas Ciclina-Dependientes/genética , Genes/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Quinasa 9 Dependiente de la Ciclina , ADN/química , ADN/genética , Exones , Células HL-60 , Células HeLa , Humanos , Intrones , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Transcripción Genética

RESUMEN

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.

Asunto(s)

Ciclinas/genética , Cisteína/genética , Productos del Gen tat/metabolismo , VIH-1/genética , Zinc/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Clonación Molecular , Secuencia Conservada/genética , Ciclina T , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regulación de la Expresión Génica/genética , Duplicado del Terminal Largo de VIH/genética , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis/genética , ARN Viral/genética , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido , Activación Transcripcional/genética , Proteínas Virales/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

TAK, a multisubunit cellular protein kinase that specifically associates with the human immunodeficiency virus Tat proteins and hyperphosphorylates the carboxyl-terminal domain of RNA polymerase II, is a cofactor for Tat and mediates its transactivation function. The catalytic subunit of TAK has been identified as cyclin-dependent kinase Cdk9, and its regulatory partner has been identified as cyclin T1; these proteins are also components of positive transcription elongation factor P-TEFb. TAK activity is up-regulated upon activation of peripheral blood lymphocytes and following macrophage differentiation of promonocytic cell lines. We have found that activation of peripheral blood lymphocytes results in increased mRNA and protein levels of both Cdk9 and cyclin T1. Cdk9 and cyclin T1 induction occurred in purified CD4(+) primary T cells activated by a variety of stimuli. In contrast, phorbol ester-induced differentiation of promonocytic cell lines into macrophage-like cells produced a large induction of cyclin T1 protein expression from nearly undetectable levels, while Cdk9 protein levels remained at a constant high level. Measurements of cyclin T1 mRNA levels in a promonocytic cell line suggested that regulation of cyclin T1 occurs at a posttranscriptional level. These results suggest that cyclin T1 and TAK function may be required in differentiated monocytes and further show that TAK activity can be regulated by distinct mechanisms in different cell types.

Asunto(s)

Productos del Gen tat/metabolismo , VIH/genética , VIH/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Diferenciación Celular , Línea Celular , Ciclina T , Quinasa 9 Dependiente de la Ciclina , Ciclinas/biosíntesis , Ciclinas/genética , Productos del Gen tat/sangre , Humanos , Activación de Linfocitos , Linfocitos/metabolismo , Linfocitos/virología , Monocitos/citología , Monocitos/metabolismo , Monocitos/virología , Factor B de Elongación Transcripcional Positiva , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/sangre , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

During transcription of mRNA genes, there is a correlation between the phosphorylation state of the C-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II) and the ability of the RNAP II complex to processively transcribe the gene. To examine the involvement of CTD phosphorylation in modulation of RNAP II function, we have analyzed the ability of a known CTD kinase, human Cdk8, to modulate HIV-1 LTR-driven gene expression upon directed targeting to a promoter-proximal nascent RNA element. The results indicated that Cdk8, when localized to an RNA element, activates gene expression in a catalysis-dependent manner. Also, Cdk8 targeted to RNA was observed to act in a synergystic manner with DNA-targeted Sp1 but not with DNA-targeted HIV-1 Tat, suggesting that RNA-targeted Cdk8 acts on similar rate limiting post-initiation events as Tat. As recent observations suggest that Tat/TAR-mediated transcription of the proviral genome of HIV depends on specific phosphorylation of RNAP II in its CTD by the Tat-associated kinase (TAK/p-TEFb/Cdk9), our results indicate that Cdk8 shares with Cdk9 the ability to modulate transcription upon targeting to a nascent RNA element.

Asunto(s)

Quinasas Ciclina-Dependientes , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/metabolismo , ARN/genética , ARN/metabolismo , Catálisis , Quinasa 8 Dependiente de Ciclina , Productos del Gen rev/genética , Productos del Gen rev/metabolismo , Productos del Gen tat/metabolismo , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción Sp1/metabolismo , Transfección

RESUMEN

The human cdc2-related kinase PITALRE is the catalytic component of TAK, the Tat-associated kinase. Previously, we have proposed that TAK is a cellular factor that mediates Tat transactivation function. Here we demonstrate that transient overexpression of PITALRE specifically squelches Tat-1 activation of both a transfected and an integrated human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR), suggesting that PITALRE mediates Tat function as a multiprotein complex. A catalytic mutant of PITALRE, D167N, was found to be more efficient than wild-type PITALRE in squelching Tat transactivation. Neither wild-type PITALRE nor D167N was able to squelch transactivation of the human T-cell leukemia type 1 LTR by the Tax protein. Additionally, we show that artificial targeting of PITALRE to a nascent RNA element, in the absence of Tat, activated HIV-1 LTR expression. These results indicate that a PITALRE-containing complex mediates transactivation by Tat and suggest that Tat proteins function by localizing such a PITALRE-containing complex to the site of the transcribing provirus.

Asunto(s)

Productos del Gen tat/genética , VIH-1/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Activación Transcripcional , Fusión Artificial Génica , Catálisis , Quinasa 9 Dependiente de la Ciclina , Expresión Génica , Genes rev , Duplicado del Terminal Largo de VIH , Células HeLa , Humanos , Mutagénesis , Factor B de Elongación Transcripcional Positiva , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Integración Viral , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612-1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

Asunto(s)

Quinasas Ciclina-Dependientes/metabolismo , Infecciones por VIH/enzimología , VIH-1/enzimología , Linfocitos/virología , Monocitos/virología , Proteínas Serina-Treonina Quinasas/metabolismo , Diferenciación Celular , Quinasas Ciclina-Dependientes/genética , Inducción Enzimática , Productos del Gen tat , Infecciones por VIH/patología , Infecciones por VIH/virología , Humanos , Activación de Linfocitos , Linfocitos/enzimología , Linfocitos/patología , Monocitos/patología , Factor B de Elongación Transcripcional Positiva , Proteínas Serina-Treonina Quinasas/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

The Tat proteins of human immunodeficiency virus types 1 (HIV-1) and 2 (HIV-2), termed Tat-1 and Tat-2, respectively, are essential for efficient viral replication. Tat proteins activate viral transcription by binding to the TAR RNA stem-loop structure at the 5' end of viral transcripts. We used an in vitro selection procedure to identify RNAs present in a large sequence pool that are able to bind to purified Tat-2 protein. The sequences of the selected RNAs demonstrated a consensus feature: 20 of 27 RNAs contained computer-predicted loop structures that were >50% U or C nucleotides. A selected RNA was characterized for its in vitro binding properties to various Tat-2 proteins. This synthetic RNA was bound by wild-type Tat-2 proteins with an affinity that was only slightly lower than that of the natural HIV-2 TAR RNA. Tat-2 required a wild-type RNA binding domain to bind to this synthetic RNA. This study indicates that in vitro selection techniques can be used to investigate Tat protein-TAR RNA interactions. Copyright 1997 S. Karger AG, Basel

RESUMEN

Previously, we showed that the viral transactivator proteins E1A and VP16 specifically interact with a cellular CTD kinase activity in vitro. We now report that E1A and VP16 complexes contain human CDK8, a newly identified member of the cyclin-dependent kinase family that has been shown to be a component of the RNA polymerase II (RNAP II) holoenzyme complex. The presence of CDK8 in the E1A- and VP16-containing complexes is specific for a functional activation domain of these viral transactivators, strongly suggesting that this association is relevant for the transactivation function of E1A and VP16. We show that CDK8 is associated with CTD kinase activity and that CDK8 co-fractionates with E1A- and VP16-associated CTD kinase activity over several chromatography columns. Therefore, CDK8 is likely responsible for the E1A- and VP16-associated CTD kinase activity. Gel filtration chromatography indicates that the E1A- and VP16-associated CTD kinase activity has a molecular size of approximately 1.5 MDa and contains cyclin C and the human homolog of SRB7 in addition to CDK8. This implies that E1A and VP16 associate with the RNAP II holoenyzme. We also looked at the transcriptional activity of CDK8 and found that CDK8 can function as a transcriptional activator when fused to the DNA binding domain of GAL4. Surprisingly, the ability of GAL4-CDK8 to activate transcription in this assay was not dependent on the kinase activity of CDK8, since a kinase-deficient mutant of CDK8 stimulated transcription nearly as well as wild-type GAL4-CDK8. This suggests that CDK8 may play a role in transcription that is distinct from its ability to function as a CTD kinase.

Asunto(s)

Proteínas E1A de Adenovirus/metabolismo , Quinasas Ciclina-Dependientes , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ciclina C , Quinasa 8 Dependiente de Ciclina , Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Factores de Transcripción/metabolismo , Transcripción Genética

RESUMEN

Human immunodeficiency virus types 1 and 2 encode closely related proteins, Tat-1 and Tat-2, that stimulate viral transcription. Previously, we showed that the activation domains of these proteins specifically interact in vitro with a cellular protein kinase named TAK. In vitro, TAK phosphorylates the Tat-2 but not the Tat-1 protein, a 42-kDa polypeptide of unknown identity, and the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP II). We now show that the 42-kDa substrate of TAK cochromatographs with TAK activity, suggesting that this 42-kDa polypeptide is a subunit of TAK. We also show that the Tat proteins specifically associate with TAK in vivo, since wild-type Tat-1 and Tat-2 proteins expressed in mammalian cells, but not mutant Tat proteins containing a nonfunctional activation domain, can be coimmunoprecipitated with TAK. We also mapped the in vivo phosphorylation sites of Tat-2 to the carboxyl terminus of the protein, but analysis of proteins with mutations at these sites suggests that phosphorylation is not essential for Tat-2 transactivation function. We further investigated whether the CTD of RNAP II is required for Tat function in vivo. Using plasmid constructs that express an alpha-amanitin-resistant RNAP II subunit with a truncated or full-length CTD, we found that an intact CTD is required for Tat function. These observations strengthen the proposal that the mechanism of action of Tat involves the recruitment or activation of TAK, resulting in activated transcription through phosphorylation of the CTD.

Asunto(s)

Productos del Gen tat/metabolismo , VIH-1/metabolismo , VIH-2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Animales , Sitios de Unión , Línea Celular Transformada , Chlorocebus aethiops , Células HeLa , Humanos , Fosforilación , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

SUMMARY: Tat regulates human immunodeficiency virus type 1 (HIV-1) gene expression by increasing both the rate of transcription initiation and the efficiency of transcription elongation. The ability of Tat to facilitate HIV-1 transcription preinitiation complex formation suggests that components of the basal transcriptional machinery may be targeted by Tat. Previous studies have demonstrated that Tat interacts directly with the human TATA-binding protein (TBP) and specific TBP-associated factors (TAFS) that comprise the TFIID complex. Here, in vitro glutathione S-transferase protein binding assays containing fully functional or transactivation-defective mutant Tat proteins have been used to investigate the functional significance of the direct interaction between Tat and TBP relative to Tat transactivation. Results demonstrate that full-length Tat, as well as the activation domain of Tat alone, binds human TBP in vitro. Site-directed mutations within the activation domain of Tat (C22G and P18IS) that abrogate transactivation by Tat in vivo fail to inhibit Tat-TBP binding. Full-length Tat, the activation domain of Tat alone, and a transactivation-defective mutant of Tat that lacks N-terminal amino acid residues 2-36 bind with equal efficiencies to TBP provided that the H1 alpha helical domain that maps to amino acids 167-220 within the highly conserved carboxyl terminus of TBP is maintained. These data indicate that an activity mapped within the activation domain of Tat, which is distinct from Tat-TBP binding. is required for transactivation by Tat.

Asunto(s)

Proteínas de Unión al ADN/genética , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , VIH-1/genética , VIH-1/metabolismo , Mutación , Factores de Transcripción/genética , Sitios de Unión/genética , Regulación Viral de la Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Humanos , Técnicas In Vitro , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Proteína de Unión a TATA-Box , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II has been implicated as an important step in transcriptional regulation. Previously, we reported that a cellular CTD kinase, TAK, is targeted by the human immunodeficiency virus transactivator Tat. In the present study, we analyzed several other transactivators for the ability to interact with CTD kinases in vitro. The adenovirus E1A and herpes simplex virus VP16 proteins, but not other transactivators tested, were found to associate with a cellular kinase activity that hyperphosphorylates the CTD. The interaction is dependent upon a functional activation domain of E1A or VP16, suggesting that the interaction with a CTD kinase is relevant for the transactivation function of these proteins. The CTD kinase activities that interact with E1A and VP16 are related to each other but distinct from TAK. The Tat-, E1A- and VP16-associated CTD kinase activities detected in our assay also appear unrelated to MO15, the catalytic component of the CTD kinase activity of the general transcription factor TFIIH. Thus, this study has identified a novel interaction between viral transactivators and a cellular CTD kinase and suggests that at least two CTD kinases may mediate responses to viral transactivators.

Asunto(s)

Proteínas E1A de Adenovirus/metabolismo , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Proteínas Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Transactivadores/metabolismo , Secuencia de Bases , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación

RESUMEN

A double transdominant fusion gene (trev) designed to inhibit two essential HIV functions simultaneously was constructed by linking tat and rev transdominant mutants. Trev independently inhibited both Tat and Rev functions, localized within the nucleus and cells transfected with trev showed a stable inhibition of HIV-1-mediated cytopathicity. A retroviral vector of trev was made and shown also to confer protection from HIV cytopathic effects. Simultaneous inhibition of two essential viral genes presents significant advantages for potential gene therapy treatment of HIV infection over conventional single effect molecules.

Asunto(s)

Clonación Molecular , Genes rev , Genes tat , VIH-1/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Efecto Citopatogénico Viral , Productos del Gen rev/fisiología , Productos del Gen tat/fisiología , Genes Dominantes/genética , Vectores Genéticos/genética , Humanos , Luciferasas/genética , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Transfección , Replicación Viral , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Efficient replication of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) requires the virus transactivator proteins known as Tat. In order to understand the molecular mechanisms involved in Tat transactivation, it is essential to identify the cellular target(s) of the Tat activation domain. Using an in vitro kinase assay, we previously identified a cellular protein kinase activity, Tat-associated kinase (TAK), that specifically binds to the activation domains of Tat proteins. Here it is demonstrated that TAK fulfills the genetic criteria established for a Tat cofactor. TAK binds in vitro to the activation domains of the Tat proteins of HIV-1 and HIV-2 and the distantly related lentivirus equine infectious anemia virus but not to mutant Tat proteins that contain nonfunctional activation domains. In addition, it is shown that TAK is sensitive to dichloro-1-beta-D-ribofuranosylbenzimidazole, a nucleoside analog that inhibits a limited number of kinases and is known to inhibit Tat transactivation in vivo and in vitro. We have further identified an in vitro substrate of TAK, the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation of the carboxyl-terminal domain has been proposed to trigger the transition from initiation to active elongation and also to influence later stages during elongation. Taken together, these results imply that TAK is a very promising candidate for a cellular factor that mediates Tat transactivation.

Asunto(s)

Regulación Viral de la Expresión Génica , Productos del Gen tat/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/metabolismo , Núcleo Celular/enzimología , Diclororribofuranosil Benzoimidazol/farmacología , VIH-1/metabolismo , VIH-2/metabolismo , Células HeLa/enzimología , Humanos , Fosforilación , Factor B de Elongación Transcripcional Positiva , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transducción de Señal , Transcripción Genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Each of the two stem-loop structures in the HIV-2 TAR (TAR-2) RNA element contains a dinucleotide bulge that specifies a binding site in vitro for the HIV-2 Tat transactivator protein. A TAR-2 RNA with both bulges deleted is very weakly transactivated in vivo by the HIV-2 Tat protein. To gain insight into general features of Tat protein:TAR RNA interactions, we have analyzed the significance of the dinucleotide bulges in TAR-2 RNA for in vitro binding and in vivo transactivation by the related HIV-1 Tat protein. The HIV-1 Tat protein has been shown previously to bind efficiently to wild-type TAR-2 RNA and fully transactivates the HIV-2 LTR. We found that the 5' proximal bulge and the 3' distal bulge appear to specify a high and low affinity binding site in vitro, respectively, for the HIV-1 Tat protein. Wild-type TAR-2 RNA was found to be able to bind HIV-1 Tat proteins simultaneously at each bulge binding site in vitro. A TAR-2 RNA with both bulges deleted was greatly defective for in vitro binding by the HIV-1 Tat protein. Surprisingly, the TAR-2 RNA with both bulges deleted was efficiently transactivated in vivo by the HIV-1 Tat protein, indicating that the HIV-1 Tat protein (but not HIV-2 Tat protein) is able to strongly activate transcription of a TAR RNA with no apparent bulge binding site.

Asunto(s)

Productos del Gen tat/fisiología , Duplicado del Terminal Largo de VIH , VIH-2/genética , ARN Viral/genética , Activación Transcripcional , Secuencia de Bases , Sitios de Unión , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleótidos/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 encode related transcriptional activators known as Tat-1 and Tat-2, respectively, that are required for efficient viral replication. The Tat proteins have been studied extensively, and it appears that their mechanism of action is unique to the primate immunodeficiency viruses or a few distantly related lentiviruses. Here we describe a collection of 24 wild-type and mutant Tat-1 and Tat-2 proteins that are expressed in Escherichia coli as fusions with glutathione S-transferase (GST). The GST-Tat fusions can be used for biochemical studies after simple purification from E. coli lysates in a single step under nondenaturing conditions. The availability of these GST-Tat fusions should be useful to investigators examining biochemical properties of Tat-1 and Tat-2 proteins. E. coli cultures harboring GST-Tat fusions described here are available through the National Institute of Health AIDS Research and Reference Reagent Program.

Asunto(s)

Productos del Gen tat/fisiología , Glutatión Transferasa/metabolismo , VIH-1/química , VIH-2/química , Proteínas Recombinantes de Fusión/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Exones , Expresión Génica , Productos del Gen tat/química , Productos del Gen tat/genética , Genes tat , Vectores Genéticos , VIH-1/genética , VIH-2/genética , Humanos , Datos de Secuencia Molecular , Plásmidos , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) express related Tat proteins that are encoded in two exons. Tat proteins bind directly to the TAR RNA element contained in the 5' ends of viral transcripts and thereby stimulate transcription through an as yet unidentified mechanism. We have investigated the functional significance of exon2 of the HIV-2 Tat protein by examining properties of proteins consisting of exon1 alone or exon1 + 2. In transactivation assays in vivo, exon2 modestly increased HIV-2 Tat stimulation of transcription from the HIV-2 long terminal repeat (LTR) but had no effect on transcription from the HIV-1 LTR. In HeLa cells, exon2 increased transactivation of the HIV-2 LTR by approximately three-fold, while in COS and Jurkat cells this value was less than two-fold. In binding assays in vitro, exon2 increased the binding affinity of the HIV-2 Tat protein to HIV-2 TAR RNA. Results with GAL4 fusion proteins and a synthetic promoter containing GAL4 DNA binding sites indicated that exon2 does not contribute to the HIV-2 Tat activation domain. These observations suggest that exon2 of HIV-2 Tat contributes to transactivation of the HIV-2 LTR by increasing the binding affinity to HIV-2 TAR RNA.

Asunto(s)

Exones , Productos del Gen tat/genética , Duplicado del Terminal Largo de VIH/fisiología , VIH-2/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional , Sitios de Unión , ADN/metabolismo , Proteínas de Unión al ADN , Proteínas Fúngicas/genética , Productos del Gen tat/metabolismo , VIH-1/genética , VIH-2/química , Células HeLa , Humanos , Técnicas de Inmunoadsorción , Regiones Promotoras Genéticas , Sondas ARN , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) encode related proteins called Tat-1 and Tat-2, respectively, that bind directly to the TAR RNA element contained at the 5' ends of viral transcripts and thereby stimulate transcription through an as yet unidentified mechanism. The determinants in the HIV-1 TAR element (TAR-1) that specify binding by the Tat-1 protein have been extensively characterized, while little is known about determinants in the HIV-2 TAR element (TAR-2) that specify binding by the Tat-2 protein. The HIV-2 TAR RNA element (TAR-2) is known to be composed of two stem-loop structures. A dinucleotide bulge is found in each stem of TAR-2 RNA, analogous to the crucial trinucleotide bulge in the single stem-loop of HIV-1 TAR RNA that is the primary binding determinant for binding by the HIV-1 Tat protein. Our results of a nuclease digestion analysis demonstrated that the 5' proximal bulge in TAR-2 is significantly less sensitive to digestion by single-strand specific nucleases than the 3' distal bulge, suggesting that the 5' bulge may be involved in tertiary interaction with other regions of TAR RNA. Deletion of both bulges reduced binding in vitro by the Tat-2 protein and largely abolished transactivation in vivo by Tat-2. Deletion of either bulge alone simplified the pattern of protein/RNA complexes in a gel shift assay, but did not reduce the overall binding affinity of Tat-2. Deletion of the 5' bulge reduced Tat-2 transactivation in vivo to a level approximately 30% that of wild-type TAR-2, while deletion of the 3' bulge had no measurable effect in vivo. Our results suggest that each dinucleotide bulge specifies a Tat-2 binding site, but in the wild-type TAR-2 element the 3' bulge binding site does not appear to be utilized in vivo.

Asunto(s)

VIH-2/genética , ARN Viral/química , Proteínas de Unión al ARN/genética , Secuencia de Bases , Sitios de Unión , ADN Viral , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas de Unión al ARN/química , Eliminación de Secuencia , Activación Transcripcional