RESUMEN
Human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export of viral RNAs involves the interaction of its leucine-rich nuclear export sequence (NES) with nuclear cofactors. In yeast two-hybrid screens of a human lymph node derived cDNA expression library, we identified the human nucleoporin Nup98 as a highly specific and potent interactor of the Rev NES. Using an extensive panel of nuclear export positive and negative mutants of the functionally homologous NESs of the HIV-1 Rev, human T cell leukemia virus type 1 (HTLV-1) Rex, and equine infectious anemia virus (EIAV) Rev proteins, physiologically significant interaction of hNup98 with the various NESs was demonstrated. Missense mutations in the yeast nuclear export factor Crm1p that abrogated Rev NES interaction with the XXFG repeat-containing nucleoporin, Rab/hRIP, had minimal effects on the interaction of GLFG repeat-containing hNup98. Functional analysis of Nup98 domains required for nuclear localization demonstrated that the entire ORF was required for efficient incorporation into the nuclear envelope. A putative nuclear localization signal was identified downstream of the GLFG repeat region. Whereas overexpression of both full-length Nup98 and the amino-terminal GLFG repeat region, but not the unique carboxy-terminal region, induced significant suppression of HIV unspliced RNA export, lower levels of exogenous Nup98 expression resulted in a relatively modest increase in unspliced RNA export. These results suggest a physiological role for hNup98 in modulating Rev-dependent RNA export during HIV infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Productos del Gen rev/metabolismo , VIH-1/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Línea Celular , Núcleo Celular/metabolismo , Células HeLa , Humanos , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos Híbridos , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.
Asunto(s)
Transporte Activo de Núcleo Celular , Productos del Gen rev/metabolismo , Cinesinas/metabolismo , Leucina/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Cinesinas/química , Cinesinas/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismoRESUMEN
The Bcl-2 homology 3 (BH3) domain is present in most members of the Bcl-2 protein family and is required to confer the death-inducing properties of pro-apoptotic members, including Bax, Bak, Bad, and Bik, in cell-based assay systems. To determine whether the BH3 domain possesses a similar role in tumor tissues in vivo, we overexpressed the wild-type Bik protein and its BH3-deleted counterpart, using adenoviral technology, in chemoresistant human tumor prostate (PC-3) and colon (HT-29) cell lines growing in vitro and in vivo. Bik caused apoptosis in both PC-3 and HT-29 cells in vitro by inducing the release of cytochrome c from mitochondria to cytoplasm, resulting in the catalytic activation of caspases 9, 7, and 3 and cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. When the BH3 domain was deleted from the Bik protein, no effect on mitochondrial activity or cell morphology could be observed. Furthermore, intratumoral injection of an adenovirus vector expressing the Bik gene, but not the deleted BH3 Bik gene, suppressed the growth of PC-3 and HT-29 xenografts established in nude mice. Histological examination of tumors from mice treated with the wild-type Bik adenoviral construct demonstrated cellular debris, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive staining, and morphological changes associated with apoptosis. In contrast, tissue sections obtained from tumors treated with the BH3-deleted Bik adenoviral construct showed no evidence of apoptosis. Thus, our results suggest that the BH3 domain is required for the antitumor activity of the Bik protein and provides a novel therapeutic approach for cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas de la Membrana , Proteínas/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Adenoviridae/genética , Animales , Proteínas Reguladoras de la Apoptosis , Western Blotting , Caspasas/biosíntesis , Caspasas/genética , División Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Grupo Citocromo c/metabolismo , Femenino , Vectores Genéticos , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Estructura Terciaria de Proteína , Proteínas/genética , Células Tumorales Cultivadas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have investigated the role of mitochondrial Ca(2+) (Ca(m)) homeostasis in cell survival. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) preceded activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 led to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik led to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induced a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also led to apoptosis. We suggest that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, our results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival.
Asunto(s)
Calcio/metabolismo , Homeostasis , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Humanos , Potenciales de la Membrana , Mitocondrias/fisiología , Células Tumorales CultivadasRESUMEN
Adenovirus E1B 19 kDa protein protects against cell death induced by viral infection and certain external stimuli. The Bcl-2 protein can functionally substitute for the E1B 19 kDa protein. To identify cellular targets for the 19 kDa protein, we used the two-hybrid screen in yeast. We have isolated cDNAs for three different proteins, designated Nip1, Nip2, and Nip3, that interact with the 19 kDa protein. Mutational analysis indicates that these proteins do not associate with 19 kDa mutants defective in suppression of cell death, suggesting a correlation between interaction of these proteins and suppression of cell death. These proteins also associate with discrete sequence motifs in the Bcl-2 protein that are homologous to motifs of the 19 kDa protein. Our results suggest that two diverse proteins, the E1B 19 kDa and the Bcl-2 proteins, promote cell survival through interaction with a common set of cellular proteins.
Asunto(s)
Proteínas E1B de Adenovirus/fisiología , Proteínas de Unión al Calcio/fisiología , Proteínas Portadoras , Muerte Celular , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Supresoras de Tumor , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/genética , Compartimento Celular , Clonación Molecular , Análisis Mutacional de ADN , Células HeLa , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Membrana Nuclear/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2 , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The bel1 gene of human spumaretrovirus (HSRV) encodes a 300-amino-acid nuclear protein termed Bel1 that is a potent activator of transcription from the cognate long terminal repeat (LTR). Bel1 can also efficiently activate the human immunodeficiency virus type 1 (HIV-1) LTR. We have previously shown that the amino-terminal 227-residue region (minimal activator region) of Bel1 can activate the HSRV LTR at low levels and that two distinct domains within the carboxy-terminal 73 residues, from residues 255 to 266 and 272 to 300, that bear little sequence homology can independently enhance the activity of the minimal activator domain (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnadurai, J. Virol. 67:161-169, 1993). We now report on the further characterization of these two transcriptional enhancement regions. Mutational analysis of the region comprising residues 255 to 266 indicates that a cluster of leucine residues is critical to the function of this region. Also, residues 273 to 287, which are identical in sequence to a 15-amino-acid segment near the carboxy terminus of the simian foamy virus transcriptional activator Taf, can independently enhance the activity of the minimal activator region. To delineate the region(s) of Bel1 that could function autonomously as an activator domain, we tested the activity of chimeric proteins comprising either wild-type or functionally defective forms of Bel1 fused to the DNA binding domain, Gal4(1-147), of the yeast transcriptional activator Gal4 on a synthetic promoter comprising Gal4 DNA binding sites linked to the adenovirus E1B TATA box (minimal promoter). Gal4-Bel1 was found to activate basal transcription from the E1B TATA box at least 35-fold, and the region responsible for this activation function was localized to the carboxy-terminal 73 amino acids. When the transcriptional enhancement regions were tested for autonomous activator function as Gal4(1-147) chimeras, residues 272 to 300, but not 255 to 266, were found to activate transcription efficiently when targeted to the E1B TATA motif and also to HSRV and HIV-1 LTRs. The highly conserved region between amino acids 273 and 287 alone was found to activate transcription efficiently when targeted to the HSRV LTR but not to the E1B TATA box or the HIV-1 LTR. Thus, our results demonstrate that the carboxy-terminal 29-amino-acid region (residues 272 to 300) contributes to Bel1 transactivation by functioning as an autonomous activator of TATA motif-directed transcription in a manner similar to that of other modular transcriptional activators.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de los Retroviridae/química , Spumavirus/genética , Transactivadores , Transactivadores/química , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Regulación Viral de la Expresión Génica , Genes Virales , VIH-1/genética , Técnicas In Vitro , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas de los Retroviridae/genética , Relación Estructura-Actividad , Transactivadores/genética , Transfección , Proteínas Estructurales Virales/genéticaRESUMEN
The bel1 gene of human spumaretrovirus (HSRV) codes for a 300-amino-acid nuclear protein, termed Bel1, that can strongly activate transcription from the cognate long terminal repeat (LTR) by at least 200-fold. Bel1 can also activate human immunodeficiency virus type 1 (HIV-1) LTR expression. By using site-directed mutagenesis, we have identified distinct regions of Bel1 essential for HSRV LTR activation. The amino-terminal 55 residues, which comprise a highly acidic region followed by a short basic stretch, were dispensable for activation. The distribution of functionally defective mutants indicates that two distinct regions between residues 56 and 300 cooperate to confer full activator function. The larger, more amino-terminal region between residues 56 and 227 is sufficient to minimally activate the HSRV LTR. It contains a region between residues 88 and 110 that is strongly conserved between the simian and human spumavirus transactivators but otherwise lacks obvious homology to known transcriptional activators except for an Arg-rich nuclear localization sequence (NLS) between residues 211 and 225 that can be functionally substituted for by the NLS of the simian virus 40 large T antigen. The carboxy-terminal 73 residues contain two functionally redundant regions that can independently augment the activity of the more N-terminal minimal activator domain by 30- to 90-fold. Comparative analysis of the effect of Bel1 mutations on HSRV and HIV-1 LTR expression revealed a similar requirement of Bel1 domains for activation of the two LTRs. Bel1 is phosphorylated in vivo, and a nuclear localization-defective mutant lacking residues 211 to 222 was severely defective for phosphorylation, whereas various deletion mutations in residues 228 to 300 resulted in a four- to eightfold reduction in phosphate incorporation. When functionally defective bel1 mutants were examined for a dominant-negative phenotype, only mutants lacking a proline-rich basic region between residues 194 and 200 or the NLS between residues 211 and 222 that were found to occupy predominantly nuclear and cytoplasmic locations, respectively, could suppress wild-type Bel1 function efficiently. In identifying two classes of dominant-negative mutants with distinct subcellular localization phenotypes, the mutational analysis of Bel1 has revealed a feature unusual for known transcriptional activators.
Asunto(s)
Genes Virales/genética , Spumavirus/genética , Secuencia de Aminoácidos , Animales , Compartimento Celular , Línea Celular , Análisis Mutacional de ADN , Genes Dominantes/genética , VIH-1/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fenotipo , Fosforilación , Señales de Clasificación de Proteína/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Relación Estructura-Actividad , Transcripción Genética , Activación Transcripcional , TransfecciónRESUMEN
The human spumaretrovirus (HSRV) genome contains, in addition to coding information for the structural proteins, open reading frames (ORFs) for at least three additional genes termed bel1, bel2 and bel3. We report here the localization of the transcriptional activator of HSRV to the bel1 ORF. In reporter-based transient expression assays in COS cells utilizing the bacterial CAT gene linked to HSRV LTR sequences between -710 and +309 with respect to the transcriptional initiation site, co-expression of the bel1 gene product alone caused an over 100 to 300-fold increase in the level of LTR activity. High-level trans-activation by bel1 was specific for the HSRV LTR, as relatively minor positive and negative regulatory effects were observed on HIV-1 LTR and RSV LTR expression, respectively, whereas HTLV-1 LTR activity remained unaffected. Distinct regions of the HSRV LTR were found to be involved in bel1-induced trans-activation. Specifically, deletions between -500 and -389 and between -136 and -62 in the U3 region resulted in a 4- and 30 to 35-fold decline, respectively, in the response to bel1. Limited mutagenesis of the bel1 ORF indicated that most of the bel1 coding region, except for the carboxy-terminal 27 residues, is essential for the activation function.
Asunto(s)
Sistemas de Lectura Abierta/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Spumavirus/genética , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Línea Celular Transformada , Deleción Cromosómica , Regulación Viral de la Expresión Génica/genética , Haplorrinos , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Activación TranscripcionalRESUMEN
The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.
Asunto(s)
Productos del Gen tat/genética , Vectores Genéticos , Infecciones por VIH/terapia , VIH-1/genética , Adenovirus Humanos/genética , Supervivencia Celular , Clonación Molecular , Expresión Génica , Productos del Gen rev/genética , Células HeLa , Técnicas In Vitro , ARN Mensajero/genética , Timidina Quinasa/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
The rev protein (Rev) of human immunodeficiency virus increases the cytoplasmic expression of viral structural gene mRNAs. We had previously reported the existence of a region (residues 73-98) near the carboxy-terminus in HIV-1 Rev essential for its function. To further define the structural elements in this region, we examined the effects of substitution mutations in highly conserved residues in this region, between amino acids 75-81, on Rev function. Mutations in Pro76-77 and Arg80 retained Rev function, whereas those in Leu75 and Leu81 abolished Rev activity and exhibited trans-dominant suppression of wt Rev function. The Leu81 mutation, in particular, exhibited an efficient dominant negative phenotype. Leu75 and Leu81 thus appear to define residues essential to the Rev "effector" function.
Asunto(s)
Regulación Viral de la Expresión Génica , Productos del Gen rev/genética , VIH-1/genética , Mutación , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/análisis , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.
Asunto(s)
Genes Virales , Genes rev , VIH-1/genética , Activación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Quimera , Cloranfenicol O-Acetiltransferasa , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Transfección , beta-GalactosidasaRESUMEN
We have studied the effect of template replication on transcriptional activation of the promoters for the adenoviral protein IX and E1a genes using a short-term transient expression assay. DNA replication mediated modulation of the transcriptional activity of these promoters was monitored using plasmids with limited replication capability conferred by the SV40 minimal origin of DNA replication in the monkey kidney cell line, COS M6. Our results indicate a substantial increase in protein IX promoter directed transcription in a dosage-independent manner as a consequence of the DNA replication process. In contrast, the transcriptional activity of the adenovirus 2 E1a promoter was not significantly altered. In addition to this DNA replication-mediated effect, transcription from the protein IX promoter was positively regulated by the adenovirus E1a 13S and E4 gene products but was repressed by the E1a 12S gene product to a limited extent. The evidence suggests that the effect of template replication on the transcriptional activity of plasmid-borne protein IX and E1a gene promoters in this transient expression system closely mimics the situation on the viral chromosome.