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1.
Oncogene ; 34(4): 496-505, 2015 Jan 22.
Article in English | MEDLINE | ID: mdl-24469036

ABSTRACT

Many oncogenic viruses activate nuclear factor-κB (NF-κB) as a part of their replicative cycles. We have shown recently that persistent and potentially oncogenic activation of NF-κB by the human T-lymphotropic virus 1 (HTLV-1) oncoprotein Tax immediately triggers a host senescence response mediated by cyclin-dependent kinase inhibitors: p21(CIP1/WAF1) (p21) and p27(Kip1) (p27) Here we demonstrate that RelA/NF-κB activation by Kaposi sarcoma herpesvirus (KSHV) latency protein vFLIP also leads to p21/p27 upregulation and G1 cell cycle arrest. Remarkably, KSHV vCyclin, another latency protein coexpressed with vFLIP from a bicistronic latency-specific mRNA, was found to prevent the senescence and G1 arrest induced by HTLV-1 Tax and vFLIP, respectively. This is because of the known ability of vCyclin/cyclin-dependent kinase 6 complex to resist p21 and p27 inhibition and cause p27 degradation. In KSHV-transformed BCBL-1 cells, sustained vFLIP expression with small hairpin RNAs-mediated vCyclin depletion resulted in G1 arrest. The functional interdependence of vFLIP and vCyclin explains why they are cotranslated from the same viral mRNA. Importantly, deregulation of the G1 cyclin-dependent kinase can facilitate chronic I-κB kinases/NF-κB activation.


Subject(s)
G1 Phase Cell Cycle Checkpoints , NF-kappa B/metabolism , Viral Proteins/physiology , Cyclin-Dependent Kinase 6/physiology , Cyclin-Dependent Kinase Inhibitor p21/physiology , Cyclin-Dependent Kinase Inhibitor p27/physiology , Gene Products, tax , HeLa Cells , Humans , I-kappa B Kinase/physiology
2.
Clin Exp Immunol ; 175(1): 92-103, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24116893

ABSTRACT

Retroviral co-infections with human immunodeficiency virus type-1 (HIV-1) and human T cell leukaemia virus type 1 (HTLV-1) or type 2 (HTLV-2) are prevalent in many areas worldwide. It has been observed that HIV-1/HTLV-2 co-infections are associated with slower rates of CD4(+) T cell decline and delayed progression to AIDS. This immunological benefit has been linked to the ability of Tax2, the transcriptional activating protein of HTLV-2, to induce the expression of macrophage inflammatory protein (MIP)-1α/CCL3, MIP-1ß/CCL4 and regulated upon activation normal T cell expressed and secreted (RANTES)/CCL5 and to down-regulate the expression of the CCR5 co-receptor in peripheral blood mononuclear cells (PBMCs). This study aimed to assess the role of Tax2-mediated activation of the nuclear factor kappa B (NF-κB) signalling pathway on the production of the anti-viral CC-chemokines MIP-1α, MIP-1ß and RANTES. Recombinant Tax1 and Tax2 proteins, or proteins expressed via adenoviral vectors used to infect cells, were tested for their ability to activate the NF-κB pathway in cultured PBMCs in the presence or absence of NF-κB pathway inhibitors. Results showed a significant release of MIP-1α, MIP-1ß and RANTES by PBMCs after the activation of p65/RelA and p50. The secretion of these CC-chemokines was significantly reduced (P < 0·05) by canonical NF-κB signalling inhibitors. In conclusion, Tax2 protein may promote innate anti-viral immune responses through the activation of the canonical NF-κB pathway.


Subject(s)
Chemokines, CC/immunology , Gene Products, tax/immunology , HTLV-II Infections/immunology , Human T-lymphotropic virus 2/immunology , Leukocytes, Mononuclear/immunology , NF-kappa B p50 Subunit/immunology , Signal Transduction/immunology , Transcription Factor RelA/immunology , Cell Line , Female , Gene Expression Regulation/immunology , HTLV-II Infections/pathology , Humans , Immunity, Innate , Leukocytes, Mononuclear/pathology , Leukocytes, Mononuclear/virology , Male
3.
Blood ; 97(7): 2137-44, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11264182

ABSTRACT

Human T-cell leukemia virus type I (HTLV-I) Tax is a potent transcriptional regulator that can activate or repress specific cellular genes and that has been proposed to contribute to leukemogenesis in adult T-cell leukemia. Previously, HTLV-I- infected T-cell clones were found to be resistant to growth inhibition by transforming growth factor (TGF)-beta. Here it is shown that Tax can perturb Smad-dependent TGF-beta signaling even though no direct interaction of Tax and Smad proteins could be detected. Importantly, a mutant Tax of CREB-binding protein (CBP)/p300 binding site, could not repress the Smad transactivation function, suggesting that the CBP/p300 binding domain of Tax is essential for the suppression of Smad function. Because both Tax and Smad are known to interact with CBP/p300 for the potentiation of their transcriptional activities, the effect of CBP/p300 on suppression of Smad-mediated transactivation by Tax was examined. Overexpression of CBP/p300 reversed Tax-mediated inhibition of Smad transactivation. Furthermore, Smad could repress Tax transcriptional activation, indicating reciprocal repression between Tax and Smad. These results suggest that Tax interferes with the recruitment of CBP/p300 into transcription initiation complexes on TGF-beta-responsive elements through its binding to CBP/p300. The novel function of Tax as a repressor of TGF-beta signaling may contribute to HTLV-I leukemogenesis. (Blood. 2001;97:2137-2144)


Subject(s)
Activin Receptors, Type I , DNA-Binding Proteins/physiology , Gene Products, tax/physiology , Nuclear Proteins/metabolism , Signal Transduction/drug effects , Trans-Activators/metabolism , Trans-Activators/physiology , Transforming Growth Factor beta/antagonists & inhibitors , Xenopus Proteins , Animals , Binding Sites , COS Cells , Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic/genetics , Chlorocebus aethiops , Genes, pX , Humans , Liver Neoplasms/pathology , Lung , Macromolecular Substances , Mink , Nerve Growth Factors , Nuclear Proteins/genetics , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/physiology , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/physiology , Recombinant Fusion Proteins/physiology , Regulatory Sequences, Nucleic Acid , Smad Proteins , Smad2 Protein , Smad3 Protein , Smad4 Protein , Trans-Activators/genetics , Transcriptional Activation , Transfection , Tumor Cells, Cultured , Xenopus laevis
4.
AIDS Res Hum Retroviruses ; 16(16): 1607-12, 2000 Nov 01.
Article in English | MEDLINE | ID: mdl-11080799

ABSTRACT

Partial proteolysis of HTLV-1 Tax protein has revealed the region surrounding amino acid residues (88)KVL(90) to be highly exposed. The protein sequence surrounding this region ((81)QRTSKTLKVLTPPIT(95)) bears resemblance to the kinase-inducible domain (KID, (129)SRRPSYRKILNE(140)) of CREB and is involved in recruiting transcriptional coactivators, p300 and CBP, for trans-activating the viral long terminal repeat (LTR). Data have also revealed the KID-like region to be important for Tax binding to DNA. Here we report that single (K88A, V89A, L90A) and double alanine substitutions (V89A-L90A) in the (88)KVL(90) motif attenuate the ability of Tax to activate NF-kappaB. Deletions near or spanning this motif also had the same effect. The alanine substitutions affect HTLV-1 LTR activation and NF-kappaB activation differently, with K88A and V89A mutants showing much reduced activities for HTLV LTR activation while retaining attenuated but significant NF-kappaB-activating function. In contrast, although the L90A mutant is similarly attenuated for NF-kappaB activation, it showed significant activity in LTR trans-activation. Incorporation of both V89A and L90A substitutions in a V89A-L90A double mutant further reduced NF-kappaB activation and completely abrogated LTR trans-activation. In aggregate, these results demonstrate the importance of the KID-like domain of Tax and implicate its interaction with cellular factors other than p300/CBP in NF-kappaB activation.


Subject(s)
Gene Products, tax/chemistry , NF-kappa B/biosynthesis , Phosphotransferases/metabolism , Amino Acid Motifs , Chloramphenicol O-Acetyltransferase/metabolism , Enzyme Activation , Gene Products, tax/genetics , Gene Products, tax/metabolism , HeLa Cells , Human T-lymphotropic virus 1/physiology , Humans , Luciferases/metabolism , Transcriptional Activation , Transfection
6.
J Biol Chem ; 275(16): 11852-7, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766811

ABSTRACT

The human T-cell lymphotropic virus, type (HTLV)-1 trans-activator, Tax, coordinates with cAMP-responsive element-binding protein (CREB) and the transcriptional co-activators p300/CBP on three 21-base pair repeat elements in the proviral long terminal repeat (LTR) to promote viral mRNA transcription. Recruitment of p300/CBP to the activator-enhancer complex, however, is insufficient to support Tax-dependent LTR trans-activation. Here, we report that the p300/CBP-associated factor (P/CAF) is a critical and integral component of the functional HTLV-1 activator-enhancer complex. The HTLV-1 Tax protein directly binds P/CAF in vitro and co-immunoprecipitates with this co-activator in vivo. The Tax mutants (K88A and V89A) defective for p300/CBP-binding and LTR trans-activation, retained their abilities to interact with P/CAF. The M47 mutant (L319R, L320S) protein, which has previously been shown to interact with p300/CBP, by contrast, failed to form complexes with P/CAF and is impaired in LTR trans-activation. Furthermore, LTR trans-activation by Tax is competitively inhibited by the adenoviral E1A 12S gene product, which displaces P/CAF from p300/CBP and inhibits the histone acetyltransferase activities of both P/CAF and p300/CBP. This inhibition is partially reversed by exogenously added P/CAF. These results imply that simultaneous recruitment of two distinct co-activators (p300/CBP and P/CAF) by Tax is essential for the assembly of a trans-activation competent, nucleoprotein complex.


Subject(s)
Gene Products, tax/metabolism , Human T-lymphotropic virus 1 , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Transcriptional Activation , Acetyltransferases/metabolism , Cell Cycle Proteins/metabolism , HeLa Cells , Histone Acetyltransferases , Humans , Mutagenesis , Nuclear Receptor Coactivator 3 , Terminal Repeat Sequences , Transcription Factors , Transfection , p300-CBP Transcription Factors
7.
J Biol Chem ; 273(42): 27339-46, 1998 Oct 16.
Article in English | MEDLINE | ID: mdl-9765261

ABSTRACT

The human T cell lymphotropic retrovirus type I (HTLV-I) trans-activator, Tax, interacts specifically with the basic-domain/leucine-zipper (bZip) protein, cAMP response element binding protein (CREB), bound to the viral Tax-responsive element consisting of three imperfect 21-base pair repeats, each with a cAMP response element core flanked by G/C-rich sequences. Here, the minimal CREB-bZip necessary for Tax binding is shown to be composed of amino acid residues 280-341. The Tax-CREB interaction involves an uninterrupted and extended alpha-helix in CREB that spans most of its basic domain to include amino acid residues localized to the NH2 terminus of the DNA binding region. Mutational analyses indicate that three residues, Arg284, Met291, and Glu299 unique to this region of the CREB/activating transcription factor-1 subfamily of bZip proteins, constitute the contact surface for Tax. Amino acid substitutions in these positions had little impact on CREB-bZip binding to DNA but abrogated its binding to Tax. Each of the contact residues for Tax are spaced approximately two helical turns apart on the side of the bZip helix directly opposite to that of the invariant DNA-binding residues. Molecular modeling reveals the Tax-contact residues to be near the minor groove of the G/C-rich DNA in the 21-base pair repeat. They most likely position Tax for minor groove contact with the G/C-rich sequences.


Subject(s)
Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , DNA, Viral/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Amino Acid Sequence , Binding Sites , Computer Simulation , Crystallography, X-Ray , Cyclic AMP Response Element-Binding Protein/genetics , Gene Expression Regulation, Viral , Leucine Zippers , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Structure, Secondary , Regulatory Sequences, Nucleic Acid
8.
Mol Cell Biol ; 18(9): 5052-61, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9710589

ABSTRACT

Human T-cell lymphotropic virus type 1 (HTLV-1) transcriptional activation is mediated by the viral transactivator, Tax, and three 21-bp repeats (Tax response element [TxRE]) located in the U3 region of the viral long terminal repeat (LTR). Each TxRE contains a core cyclic AMP response element (CRE) flanked by 5' G-rich and 3' C-rich sequences. The TxRE binds CREB (CRE-binding protein) and Tax to form a ternary complex and confers Tax-dependent transactivation. Recent data indicate that Tax functions as a specific link to connect CREB-binding protein (CBP)/p300 in a phosphorylation-independent manner to CREB/ATF-1 assembled on the viral 21-bp repeats. Glutathione S-transferase pull-down performed with Tax deletion mutants and peptide competition have localized the site in Tax critical for binding CBP/p300 to a highly protease-sensitive region around amino acid residues 81 to 95 (81QRTSKTLKVLTPPIT95) which lies between the domains previously proposed to be important for CREB binding and Tax subunit dimerization. Amino acid residues around the trypsin- and chymotrypsin-sensitive sites (88KVL90) of Tax bear resemblance to those in the kinase-inducible domain of CREB (129SRRPSYRKILNE140) surrounding Ser-133, which undergoes signal-induced phosphorylation to recruit CBP/p300. Site-directed mutagenesis of residues in this domain (R82A, K85A, K88A, and V89A) resulted in proteins which failed to transactivate from the HTLV-1 LTR in vivo. These mutants (K85A, K88A, and V89A) bind CREB with similar affinities as wild-type Tax, yet interaction with CBP/p300 is abrogated in various biochemical assays, indicating that the recruitment of CBP/p300 is crucial for Tax transactivation. A Tax mutant, M47, defective in the COOH-terminal transactivation domain, continued to interact with CBP/p300, suggesting that interactions with additional cellular factors are required for proper Tax function.


Subject(s)
Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Products, tax/chemistry , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Amino Acid Sequence , Binding Sites , Chloramphenicol O-Acetyltransferase/biosynthesis , Cloning, Molecular , Escherichia coli , Gene Products, tax/isolation & purification , Glutathione Transferase , HeLa Cells , Human T-lymphotropic virus 1/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Sequence Deletion , Transfection
9.
J Virol ; 72(8): 6777-84, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9658126

ABSTRACT

Human T-cell leukemia virus type 1 (HTLV-1) Tax targets I-kappaB alpha and I-kappaB beta for phosphorylation, ubiquitination, and proteasome-mediated degradation, causing the nuclear translocation of NF-kappaB/Rel proteins and transcription induction of many cellular genes. The mechanism by which a nuclear protein such as Tax stimulates I-kappaB phosphorylation and degradation remains unclear. Here, we describe two cytoplasmic mutants of Tax, designated TaxDeltaN81 and TaxDeltaN109, from which the domains important for cyclic AMP response element binding factor (CREB) and serum response factor (SRF) binding and nuclear transport have been removed. These mutants were unable to trans activate from the HTLV-1 21-bp repeats or the serum response element in the c-fos promoter. In contrast, they activated NF-kappaB reporters, suggesting that activation of NF-kappaB by Tax occurs in the cytoplasm. Incorporation of the nuclear localization signal (NLS) of the simian virus 40 large T antigen into TaxDeltaN81 and TaxDeltaN109 redirected both proteins predominantly to the nucleus yet did not restore trans activation via CREB or SRF. The NLS fusion had little effect on TaxDeltaN81 but reduced NF-kappaB trans activation by TaxDeltaN109, possibly because of its proximity to the NF-kappaB-activating domain of Tax. In contrast to wild-type Tax, the cytoplasmic TaxDeltaN mutants are not cytotoxic. Stable expression of TaxDeltaN109 in HeLa cells resulted in a significant reduction in the intracellular level of I-kappaB alpha, with the constitutive presence of NF-kappaB in the nucleus and concomitant activation of the NF-kappaB enhancer. These results are suggestive of a potential application of the TaxDeltaN109-like mutants in targeting I-kappaB degradation and NF-kappaB activation. Interestingly, a Tax species with a molecular mass similar to that of TaxDeltaN109 was identified in many HTLV-1-transformed T cells, suggesting that TaxDeltaN109-like species might play a role in HTLV-1-induced leukemogenesis.


Subject(s)
Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , NF-kappa B/metabolism , Binding Sites , Cell Line, Transformed , Chromosome Mapping , Cytoplasm/metabolism , Gene Expression , Gene Products, tax/genetics , HeLa Cells , Humans , Mutagenesis , Phenotype , Transcriptional Activation
10.
J Biol Chem ; 272(41): 25816-23, 1997 Oct 10.
Article in English | MEDLINE | ID: mdl-9325311

ABSTRACT

Tumor necrosis factor alpha (TNFalpha) has pleiotropic effects on cellular metabolism. One of the signaling paths from the TNFalpha receptor induces a stress-activated protein kinase cascade. Components within this TNFalpha kinase cascade include mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and stress-activated protein kinase/extracellular signal-regulated kinase kinase (SEK), which regulate the activity of c-Jun N-terminal kinase 1 (JNK1). Currently, molecules upstream of MEKK1 that link TNFalpha receptor to downstream kinases are not well understood. Besides TNFalpha, many other stimuli including several oncoproteins can activate JNK1. In most cases, the signaling cascade(s) leading from oncoproteins to JNK1 is poorly elucidated. We report here that the human T-cell lymphotrophic virus, type I (HTLV-I) oncoprotein, Tax, can activate JNK1. We isolated a novel human cell factor, G-protein pathway suppressor 2 (GPS2), by its ability to bind the HTLV-I oncoprotein, and we show that this factor can potently suppress Tax activation of JNK1. In trying to understand the mechanism of GPS2 activity, we found that it also suppressed TNFalpha activation of JNK1 but not TNFalpha activation of p38 kinase nor phorbol activation of extracellular signal-regulated kinase 2. Because GPS2 has minimal effect on MEKK1- or SEK-regulated JNK1 activity, it could act at a point between the TNFalpha receptor and MEKK1 in the initial step(s) of this kinase cascade. Alternatively, it is not excluded that GPS2 could work in a parallel pathway that leads from TNFalpha to JNK1. GPS2 represents a new molecule that could contribute important insights toward how cytokine- and oncoprotein-mediated signal transduction might converge.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Gene Products, tax/pharmacology , MAP Kinase Kinase Kinase 1 , Mitogen-Activated Protein Kinases , Repressor Proteins , Tumor Necrosis Factor-alpha/pharmacology , Amino Acid Sequence , Base Sequence , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Enzyme Activation , Fungal Proteins/metabolism , Gene Products, tax/metabolism , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases , Jurkat Cells , Molecular Sequence Data , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases
11.
J Invest Dermatol ; 108(6): 920-2, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9182822

ABSTRACT

The recently discovered human virus known as Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8) has been associated with body-cavity-based lymphomas in AIDS patients. It is most closely related to two other herpesviruses, the Epstein-Barr virus and herpesvirus saimiri, which are known to be associated with lymphomas in humans and nonhuman primates, respectively. To determine whether KSHV/HHV-8 is involved in the pathogenesis of mycosis fungoides (MF) and related disorders, we used a genomic PCR assay followed by confirmatory Southern blot analysis with a nested oligonucleotide probe to analyze cases for the presence of this virus. The specimens studied included fresh-frozen lesional tissues obtained from 16 patients with MF, seven with lymphomatoid papulosis, seven with primary cutaneous CD30+ large cell lymphoma of T-cell lineage, and five with Hodgkin's disease. Two T-cell tumor lines were also studied: MT4 (derived from a patient with adult T-cell leukemia/lymphoma) and Jurkat (derived from a patient with T-cell acute lymphoblastic leukemia). All cases were uniformly negative for KSHV/HHV-8, whereas Kaposi's sarcoma-positive controls and human beta-globin DNA integrity controls were appropriately positive. These findings provide strong evidence against a role for KSHV/HHV-8 in the pathogenesis of MF or associated lymphoproliferative disorders.


Subject(s)
Herpesvirus 8, Human/physiology , Hodgkin Disease/virology , Lymphoma, Large B-Cell, Diffuse/virology , Mycosis Fungoides/virology , Skin Diseases, Papulosquamous/virology , Skin Neoplasms/virology , Adult , Base Sequence , Beta-Globulins/analysis , Beta-Globulins/genetics , Beta-Globulins/metabolism , Blotting, Southern , Cells, Cultured , DNA/analysis , DNA/genetics , DNA/metabolism , DNA Primers/analysis , DNA Primers/chemistry , DNA Primers/genetics , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , DNA, Neoplasm/metabolism , DNA, Viral/analysis , DNA, Viral/chemistry , DNA, Viral/genetics , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/isolation & purification , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Humans , Jurkat Cells/chemistry , Jurkat Cells/metabolism , Jurkat Cells/pathology , Lymphoma, Large B-Cell, Diffuse/chemistry , Lymphoma, Large B-Cell, Diffuse/pathology , Mycosis Fungoides/chemistry , Mycosis Fungoides/pathology , Polymerase Chain Reaction , Skin/chemistry , Skin/metabolism , Skin/pathology , Skin Diseases, Papulosquamous/metabolism , Skin Diseases, Papulosquamous/pathology , Skin Neoplasms/chemistry , Skin Neoplasms/pathology , Tumor Cells, Cultured
12.
J Infect Dis ; 175(4): 947-50, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9086156

ABSTRACT

With the advent of AIDS, Kaposi's sarcoma (KS) has become one of the leading malignancies in sub-Saharan Africa. Recently, DNA sequences from a new human herpesvirus called KS-associated herpesvirus (KSHV) or human herpesvirus type 8 have been found in KS tumor lesions in high frequency. Analyses of tumor lesions from 38 Ugandan KS patients indicated a uniform presence of KSHV in KS tumor lesions as revealed by polymerase chain reaction and Southern hybridization. In contrast, only 31% (11/36) of the normal skin biopsies from the same patient population were positive. The frequency of KSHV DNA detection in peripheral blood mononuclear cells (PBMC) of KS patients was also high (84%, 31/37). Similar analyses revealed the presence of cytomegalovirus (21% in KS lesions) to be discordant with KS development. A large number of KS lesions (87%, 33/38) and KS PBMC (70%, 26/37) were, however, positive for Epstein-Barr virus sequences. In addition, KSHV DNA was not found in the PBMC of Ugandans without KS.


Subject(s)
Herpesviridae/isolation & purification , Herpesvirus 4, Human/isolation & purification , Leukocytes, Mononuclear/virology , Sarcoma, Kaposi/virology , Adult , DNA, Viral/analysis , Female , Humans , Male , Middle Aged , Polymerase Chain Reaction
14.
J Virol ; 70(12): 8368-74, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8970957

ABSTRACT

Human T-lymphotropic virus type 1 Tax interacts specifically with the cellular transcription factor CREB and the viral 21-bp repeat element to form a Tax-CREB-DNA ternary complex which mediates activation of viral mRNA transcription. Analyses of Tax and Tax mutants indicate that, like CREB, Tax incorporates into the ternary complex as a dimer. The ability of Tax to form a dimer is necessary for its interaction with CREB and the 21-bp element. Analyses of several Tax mutants with amino acid substitutions spanning residues 123 to 204 indicate that intersubunit Tax dimerization correlates with its ability to assemble into the ternary complex and activate transcription. Tax also enhances the DNA binding activities of specific bZip domains in vitro. The ability of Tax to enhance DNA binding of bZip proteins can be explained in part by Tax dimerization. This activity alone is not sufficient for transactivation. A dual amino acid substitution mutant of Tax, M47 (L319R, L320S), completely abrogated for activation of the human T-lymphotropic virus type 1 long terminal repeat as a result of a defect in the transactivation domain, continues to stimulate binding of bZip proteins to DNA.


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/metabolism , Repetitive Sequences, Nucleic Acid , Transcription Factors , Basic-Leucine Zipper Transcription Factors , DNA-Binding Proteins/metabolism , G-Box Binding Factors , Gene Products, tax/genetics , Human T-lymphotropic virus 1/genetics , Humans , Transcriptional Activation
15.
Virology ; 214(1): 207-14, 1995 Dec 01.
Article in English | MEDLINE | ID: mdl-8525616

ABSTRACT

The bovine leukemia virus transactivator, BLV Tax, augments transcription from three imperfect 21-bp repeats in the viral transcriptional regulatory region. Each BLV 21-bp repeat contains a cAMP response element (CRE) in the center and unique 5' and 3' neighboring sequences which are crucial for the transcriptional activation by BLV Tax. Here we describe the interactions of recombinant BLV Tax with cellular bZip proteins. The recombinant BLV Tax, tagged at the carboxy terminus with a hexahistidine extension, was prepared by solubilization in 6 M guanidine hydrochloride and renaturation on the Ni(2+)-chelating Sepharose gel matrix. The renatured BLV TaxH6 activates the BLV LTR when introduced into HeLa cells by scrape loading. Furthermore, the purified BLV TaxH6 enhances binding of members of the CREB/ATF family of bZip proteins to CRE motifs by interacting with their bZip domains in vitro. Chemical cross-linking indicates that dimerization of bZip proteins such as CREB becomes greatly facilitated in the presence of BLV Tax. These results suggest that BLV Tax interacts directly with CREB/ATF-like factors to activate viral mRNA transcription.


Subject(s)
DNA-Binding Proteins/metabolism , Gene Products, tax/metabolism , Leukemia Virus, Bovine/metabolism , Retroviridae Proteins, Oncogenic/metabolism , Trans-Activators/metabolism , Transcription Factors , Amino Acid Sequence , Animals , Base Sequence , Basic-Leucine Zipper Transcription Factors , Cattle , Cyclic AMP Response Element-Binding Protein/metabolism , DNA Primers , DNA, Viral/metabolism , G-Box Binding Factors , Humans , Leukemia Virus, Bovine/genetics , Molecular Sequence Data , Protein Binding , Repetitive Sequences, Nucleic Acid
16.
RNA ; 1(3): 317-26, 1995 May.
Article in English | MEDLINE | ID: mdl-7489503

ABSTRACT

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies.


Subject(s)
Gene Products, tax/metabolism , RNA/metabolism , Transcription Factors/metabolism , Base Sequence , Binding Sites , Binding, Competitive , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/metabolism , Gene Products, tax/antagonists & inhibitors , Molecular Sequence Data , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Protein Binding , RNA/antagonists & inhibitors , Serum Response Factor
17.
J Virol ; 69(3): 1834-41, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7853524

ABSTRACT

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-kappa B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Transcriptional Activation , Amino Acid Sequence , Base Sequence , Consensus Sequence , DNA/metabolism , DNA Primers/chemistry , Gene Expression Regulation, Viral , Gene Products, tax/chemistry , Glutathione Transferase/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Recombinant Fusion Proteins , Regulatory Sequences, Nucleic Acid , Structure-Activity Relationship
18.
Bioorg Med Chem ; 2(10): 1085-90, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7773625

ABSTRACT

Studies on the inhibition of HIV-1 protease utilizing a core isostere with replacement of the scissle bond for an alpha-amino-ketone have resulted in the development of an alpha-keto-amide isosteric replacement of the Phe-Pro scissle amide bond. The simple dipeptide isostere was shown to be a promising new core structure for the development of the enzyme inhibitors. The Ki of this core structure was determined to be 6 microM, compared to 230 microM and > 50 microM for the corresponding phosphinic acid and hydroxyethylamine isosteres.


Subject(s)
Dipeptides/chemistry , HIV Protease Inhibitors/chemistry , Amides/chemistry , Amides/pharmacology , Amines/chemistry , Amines/pharmacology , Amino Acid Sequence , Dipeptides/pharmacology , Escherichia coli , HIV Protease Inhibitors/pharmacology , Ketones/chemistry , Ketones/pharmacology , Kinetics , Molecular Sequence Data , Phenylalanine/chemistry , Proline/chemistry , Recombinant Fusion Proteins/antagonists & inhibitors
19.
J Acquir Immune Defic Syndr (1988) ; 7(7): 727-33, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8207650

ABSTRACT

To examine the effects of Mycobacterium tuberculosis on human immunodeficiency virus type 1 (HIV-1) expression, the monocytoid cell line U1 containing integrated provirus was incubated with the H37Ra strain of M. tuberculosis. This resulted in heightened expression of virus in supernatant that was partially inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha). Purified protein derivative (PPD) prepared from M. tuberculosis also could activate HIV expression, and this was less affected by anti-TNF antibody. PPD could activate the HIV promoter in both U937, the monocytoid cell line from which U1 was derived, and Jurkat, a CD4+ lymphoid line. Activation was abolished by mutations in the nuclear factor (NF)-kB binding domains. Jurkat cells transfected with a plasmid construct linking 8 NF-kB binding domains to the chloramphenicol acetyltransferase (CAT) gene showed increased activity of the reporter gene after activation with PPD. Transcriptional activation of HIV expression by mycobacteria and mycobacterial products may enhance propagation of HIV in monocytoid and lymphoid cells. This may result in accelerated HIV disease progression in persons coinfected with M. tuberculosis.


Subject(s)
Gene Expression Regulation, Viral , HIV-1/genetics , Mycobacterium tuberculosis/physiology , Tuberculin/pharmacology , Base Sequence , Cell Line , Dose-Response Relationship, Drug , Gene Expression Regulation, Viral/drug effects , HIV-1/drug effects , Humans , Molecular Sequence Data , Mutagenesis , NF-kappa B/genetics , Oligodeoxyribonucleotides/chemistry , Promoter Regions, Genetic , Repetitive Sequences, Nucleic Acid/drug effects , TATA Box , Transcription, Genetic/drug effects , Transfection , Tumor Necrosis Factor-alpha/immunology , Virus Replication/genetics
20.
Proc Natl Acad Sci U S A ; 91(12): 5642-6, 1994 Jun 07.
Article in English | MEDLINE | ID: mdl-8202541

ABSTRACT

The transactivator protein of human T-lymphotropic virus type I (HTLV-I), Tax, forms multiprotein complexes with the ubiquitous transcription factor CREB and the CREB/ATF-1 heterodimer. The interaction between Tax and CREB is highly specific and results in increased binding of the Tax/CREB complexes to the HTLV-I 21-bp repeats. Despite the extensive sequence similarities between CREB and ATF-1, Tax interacts with ATF-1 only marginally. Compared with CREB, Tax/CREB exhibits greatly increased DNA recognition specificity and preferentially assembles on a consensus binding site, GGGGG(T/A)TGACG(T/C)(A/C)TA(T/C)C-CCCC, homologous to the HTLV-I 21-bp repeats. Here we report that Tax affects CREB binding to the Tax-inducible DNA elements by interacting with the basic-leucine zipper (bZip) domain of CREB. We show by domain switching that the basic region in CREB bZip can confer on c-Jun and ATF-1 leucine zippers the ability to interact with Tax in vitro. Mutational analyses further demonstrate that the amino acid residues of CREB critical for Tax/CREB interaction are Ala-Ala-Arg at positions 282-284 (AAR284), immediately upstream of the highly conserved DNA-binding domain (R/K)XX(R/K) N(R/K)XAAXX(S/C)RX(R/K)(K/R) characteristic of all bZip proteins. Specific amino acid substitutions in AAR284 of CREB weakened or abolished Tax/CREB interaction, whereas reciprocal changes in ATF-1 allowed it to interact with Tax. These results support a model in which the specific interaction between Tax and the AAR284 residues near the DNA-binding domain of CREB results in a multiprotein complex with altered DNA recognition property. This protein complex assembles selectively on the viral Tax-responsive 21-bp repeats to augment transcription.


Subject(s)
Cyclic AMP Response Element-Binding Protein/chemistry , Gene Products, tax/chemistry , Activating Transcription Factor 1 , Amino Acid Sequence , Base Sequence , Binding Sites , Consensus Sequence , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Gene Products, tax/metabolism , Human T-lymphotropic virus 1/genetics , Humans , In Vitro Techniques , Leucine Zippers , Molecular Sequence Data , Protein Binding , Transcription Factors/metabolism
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