[Show abstract][Hide abstract] ABSTRACT: We have investigated the effects of aromatic polyamidines on HIV-1 transcription. We found a block to Tat-induced HIV-1 transcription assessed by inhibition of CAT activity in HL3T1 cells at a concentration lower than the IC50 value, suggesting that molecules with three (TAPB) and four (TAPP) benzamidine rings could be useful against HIV-1. In contrast, aromatic polyamidines with only two benzamidine rings (DAPP) did not block Tat-induced transcription. We reasoned that this effect could be due to binding of TAPB and TAPP to HIV-1 TAR RNA. By EMSA and filter binding assays, we studied possible interactions of aromatic polyamidines with HIV-1 TAR RNA. Wild-type TAR RNA or TAR RNA with mutations in the stem or bulge sequences, but retaining the stem-loop structure, was used to define the RNA-binding activities of these compounds. Our data suggest that aromatic polyamidines with two (DAPP) and four (TAPP) benzamidine rings, respectively, do not bind to TAR RNA or bind without sequence selectivity. Interestingly, an aromatic polyamidine with three benzamidine rings (TAPB) recognizes the wild-type TAR RNA in a specific manner. Furthermore, we found that introduction of one halogen atom into the benzamidine rings strongly increases the RNA-binding activity of these compounds.
Antisense and Nucleic Acid Drug Development 09/2001; 11(4):209-17.
[Show abstract][Hide abstract] ABSTRACT: Expression of the human T-cell leukemia virus type 1 (HTLV-1) oncoprotein Tax is correlated with cellular transformation contributing to the development of adult T-cell leukemia. Tax has been shown to modulate the activities of several cellular promoters. Existing evidence suggests that Tax need not directly bind to DNA to accomplish these effects but rather that it can act through binding to cellular factors, including members of the CREB/ATF family. Exact mechanisms of HTLV-1 transformation of cells have yet to be fully defined, but the process is likely to include both activation of cellular-growth-promoting factors and repression of cellular tumor-suppressing functions. While transcriptional activation has been well studied, transcriptional repression by Tax, reported recently from several studies, remains less well understood. Here, we show that Tax represses the TATA-less cyclin A promoter. Repression of the cyclin A promoter was seen in both ts13 adherent cells and Jurkat T lymphocytes. Two other TATA-less promoters, cyclin D3 and DNA polymerase alpha, were also found to be repressed by Tax. Interestingly, all three promoters share a common feature of at least one conserved upstream CREB/ATF binding site. In electrophoretic mobility shift assays, we observed that Tax altered the formation of a complex(es) at the cyclin A promoter-derived ATF site. Functionally, we correlated removal of the CREB/ATF site from the promoter with loss of repression by Tax. Furthermore, since a Tax mutant protein which binds CREB repressed the cyclin A promoter while another mutant protein which does not bind CREB did not, we propose that this Tax repression occurs through protein-protein contact with CREB/ATF.
Journal of Virology 04/2001; 75(5):2161-73. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent evidence from several investigators suggest that the human T-cell leukemia virus type 1 Tax oncoprotein represses the transcriptional activity of the tumor suppressor protein, p53. An examination of published findings reveals serious controversy as to the mechanism(s) utilized by Tax to inhibit p53 activity and whether the same mechanism is used by Tax in adherent and suspension cells. Here, we have investigated Tax-p53 interaction simultaneously in adherent epithelial (HeLa and Saos) and suspension T-lymphocyte (Jurkat) cells. Our results indicate that Tax activity through the CREB/CREB-binding protein (CBP), but not NF-kappaB, pathway is needed to repress the transcriptional activity of p53 in all tested cell lines. However, we did find that while CBP binding by Tax is necessary, it is not sufficient for inhibiting p53 function. Based on knockout cell studies, we correlated a strong genetic requirement for the ATM, but not protein kinase-dependent DNA, protein in conferring a Tax-p53-repressive phenotype.
Journal of Virology 02/2001; 75(1):396-407. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T-cell leukemia (ATL), as well as for tropical spastic paraparesis (TSP) and HTLV-I associate myelopathy (HAM). A biological understanding of the involvement of HTLV-I and in ATL has focused significantly on the workings of the virally-encoded 40 kDa phospho-oncoprotein, Tax. Tax is a transcriptional activator. Its ability to modulate the expression and function of many cellular genes has been reasoned to be a major contributory mechanism explaining HTLV-I-mediated transformation of cells. In activating cellular gene expression, Tax impinges upon several cellular signal-transduction pathways, including those for CREB/ATF and NF-kappa B. In this paper, we review aspects of Tax's transcriptional potential with particular focus on recent evidence linking Tax to IKK (I kappa B-kinase)-complex and MAP3Ks (mitogen-activated protein kinase kinase kinases).
[Show abstract][Hide abstract] ABSTRACT: Tax protein expressed by human T cell leukemia virus type 1 (HTLV-1) is a strong trans-activator of its own LTR promoter; it also affects the function of multiple cellular genes involved in cell cycle control and transcription. One way in which Tax exerts its pleiotropic effects is through protein-protein interaction with cellular cofactors. By using yeast two-hybrid technology, we have isolated several cellular proteins that bind to Tax. Two of these are MAD1, a mitotic checkpoint control protein, and TXBP151, a suppressor of tumor necrosis factor alpha-induced apoptosis. Here we discuss findings describing the role of MAD1 in exit of cells from mitosis and TXBP151 in NF-kappaB activation.
AIDS Research and Human Retroviruses 12/2000; 16(16):1633-8. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human T lymphotropic virus type 1 (HTLV-1) Tax is a multifunctional protein centrally involved in transcriptional regulation, cell cycle control, and viral transformation. The regulatory functions of Tax are thought to be mediated through protein-protein interaction with cellular cofactors. Previously we have identified several novel binding partners for Tax, including human mitotic checkpoint protein MAD1 (TXBP181), G-protein pathway suppressor GPS2 (TXBP31), and IkappaB kinase regulatory subunit IKK-gamma. Here we described two additional Tax partners, TXBP151 and TXBP121. A closer examination of the sequences of eight independent cellular Tax-binding proteins identified by us and others revealed that all of them share a single characteristic, a highly structured coiled-coil domain. We also noted that Tax and the Tax-binding coiled-coil proteins can homodimerize. Additionally, the same domain in Tax is responsible for interaction with different coiled-coil proteins. Taken together, our findings point to a particular coiled-coil structure as one of the Tax-recognition motifs. The interaction of Tax with a particular subgroup of cellular coiled-coil proteins represents one mechanism by which Tax dysregulates cell growth and proliferation.
AIDS Research and Human Retroviruses 12/2000; 16(16):1689-94. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. CCR5 is the major macrophage-tropic coreceptor for HIV-1 whereas CXC chemokine receptor 4 (CXCR4) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-CXCR4 interaction may exist. Here, we report that the HIV-1 Tat protein, which is secreted from virus-infected cells, is a CXCR4-specific antagonist. Soluble Tat selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted Tat is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.
Proceedings of the National Academy of Sciences 11/2000; 97(21):11466-71. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Expression of the human T-cell leukemia virus type I (HTLV-I) Tax oncoprotein rapidly engenders DNA damage as reflected in a significant increase of micronuclei (MN) in cells. To understand better this phenomenon, we have investigated the DNA content of MN induced by Tax. Using an approach that we termed FISHI, fluorescent in situ hybridization and incorporation, we attempted to characterize MN with centric or acentric DNA fragments for the presence or absence of free 3'-OH ends. Free 3'-OH ends were defined as those ends accessible to in situ addition of digoxigenin-dUTP using terminal deoxynucleotidyl transferase. MN were also assessed for centromeric sequences using standard fluorescent in situ hybridization (FISH). Combining these results, we determined that Tax oncoprotein increased the frequency of MN containing centric DNA with free 3'-OH and decreased the frequency of MN containing DNA fragments that had incorporation-inaccessible 3'-ends. Recently, it has been suggested that intracellular DNA breaks without detectable 3'-OH ends are stabilized by the protective addition of telomeric caps, while breaks with freely detectable 3'-OH are uncapped and are labile to degradation, incomplete replication, and loss during cell division. Accordingly, based on increased detection of free 3'-OH-containing DNA fragments, we concluded that HTLV-I Tax interferes with protective cellular mechanism(s) used normally for stabilizing DNA breaks.
Journal of Biological Chemistry 11/2000; 275(42):32906-10. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The MDR1 multidrug transporter P-gp (P-glycoprotein) is an efflux pump that extrudes diverse hydrophobic drugs and peptides from cells. Since the entry of HIV-1 into cells involves an initial interaction of the viral gp41 hydrophobic peptide with the plasma membrane, a potential effect of P-gp on HIV-1 infectivity was explored. Virus production was greatly decreased when P-gp was overexpressed at the surface of a continuous CD4(+) human T-leukemic cell line (12D7) infected with HIV-1(NL4-3), a T-tropic molecular clone of HIV-1. P-gp overexpression did not significantly alter the surface expression or distribution of either the HIV-1 receptor CD4 or the coreceptor CXCR4. Reduction of HIV-1 infectivity in P-gp-expressing cells occurred both during the fusion of viral and plasma membranes and at subsequent step(s) in the HIV-1 life cycle.
The FASEB Journal 04/2000; 14(3):516-22. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatitis C virus (HCV) is the major etiological agent of blood-borne non-A non-B hepatitis and a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. HCV core protein is a multifunctional protein with regulatory functions in cellular transcription and virus-induced transformation and pathogenesis. Here we report on the identification of a bZIP nuclear transcription protein as an HCV core cofactor for transformation. This bZIP factor, designated LZIP, activates CRE-dependent transcription and regulates cell proliferation. Loss of LZIP function in NIH 3T3 cells triggers morphological transformation and anchorage-independent growth. We show that HCV core protein aberrantly sequesters LZIP in the cytoplasm, inactivates LZIP function and potentiates cellular transformation. Our findings suggest that LZIP might serve a novel cellular tumor suppressor function that is targeted by the HCV core.
The EMBO Journal 03/2000; 19(4):729-40. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human T-cell leukemia virus type I (HTLV-I) is the etiological agent for adult T-cell leukemia (ATL) and various human myopathies/neuropathies. HTLV-I encodes a 40 kDa phosphoprotein, Tax, which has been implicated in cellular transformation. In similarity with several other oncoproteins such as Myc, Jun, and Fos, Tax is a transcriptional activator. How Tax mechanistically dysregulates the cell cycle remains unclear. Recent findings from us and others have shown that Tax targets key regulators of G1/S and M progression such as p16INK4a, cyclin D1, cyclin D3-cdk, and the mitotic spindle checkpoint apparatus. Thus, Tax influences the progression of cells in various phases of the cell cycle. In this regard, we will discuss three distinct mechanisms through which Tax affects cell-cycling: a) through direct association Tax can abrogate the inhibitory function of p16INK4a on the G1-cdks, b) Tax can also directly influence cyclin D-cdk activities by a protein-protein interaction, and c) Tax targets the HsMAD1 mitotic spindle-assembly checkpoint protein. Through these varied routes, the HTLV-I oncoprotein dysregulates cellular growth controls and engenders a proclivity of cells toward a loss of DNA-damage surveillance.
Progress in cell cycle research 02/2000; 4:157-62.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to identify and characterize human and mouse Prx-IV. We identified mouse peroxiredoxin IV (Prx-IV) by virtue of sequence homology to its human ortholog previously called AOE372. Mouse Prx-IV conserves an amino-terminal presequence coding for signal peptide. The amino acid sequences of mature mouse and human Prx-IV share 97.5% identity. Phylogenetic analysis demonstrates that Prx-IV is more closely related to Prx-I/-II/-III than to Prx-V/-VI. Previously, we mapped the mouse Prx-IV gene to chromosome X by analyzing two sets of multiloci genetic crosses. Here we performed further comparative analysis of mouse and human Prx-IV genomic loci. Consistent with the mouse results, human Prx-IV gene localized to chromosome Xp22.135-136, in close proximity to SAT and DXS7178. A bacterial artificial chromosome (BAC) clone containing the complete human Prx-IV locus was identified. The size of 7 exons and the sequences of the splice junctions were confirmed by PCR analysis. We conclude that mouse Prx-IV is abundantly expressed in many tissues. However, we could not detect Prx-IV in the conditioned media of NIH-3T3 and Jurkat cells. Mouse Prx-IV was specifically found in the nucleus-excluded region of cultured mouse cells. Intracellularly, overexpression of mouse Prx-IV prevented the production of reactive oxygen species induced by epidermal growth factor or p53. Taken together, mouse Prx-IV is likely a cytoplasmic or organellar peroxiredoxin involved in intracellular redox signaling.
Antioxidants and Redox Signaling 02/2000; 2(3):507-18. · 7.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The IE2 protein of human cytomegalovirus transactivates viral and cellular promoters through a wide variety of cis-elements, but the mechanism of its action has not been well characterized. Here, IE2-Sp1 synergy and IE2-TATA box-binding protein (TBP) interaction are examined by artificial recruitment of either Sp1 or TBP to the promoter. It was found that IE2 could cooperate with DNA-bound Sp1. A 117 amino acid glutamine-rich fragment of Sp1, which can interact with Drosophila TAF(II)110 and human TAF(II)130, was sufficient for the augmentation of IE2-driven transactivation. In binding assays in vitro, IE2 interacted directly with the C-terminal region of Sp1, which contains the zinc finger DNA-binding domain, but not with its transactivation domain, suggesting that synergy between IE2 and the transactivation domain of Sp1 might be mediated by other proteins such as TAF or TBP. It was also found that TBP recruitment to the promoter markedly increased IE2-mediated transactivation. Thus, IE2 acts synergistically with DNA-bound Sp1 and DNA-bound TBP. These results suggest that, in human cytomegalovirus IE2 transactivation, Sp1 functions at an early step such as recruitment of TBP and IE2 acts to accelerate rate-limiting steps after TBP recruitment.
Journal of General Virology 02/2000; 81(Pt 1):37-46. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Replication of the human immunodeficiency virus type 1 (HIV-1) is inhibited by interferons (IFNs), in part through activity of the IFN-inducible protein kinase PKR. To escape this antiviral effect, HIV-1 has developed strategies for blocking PKR function. We have previously shown that the HIV-1 Tat protein can associate with PKR in vitro and in vivo and inhibit PKR activity. Here we present evidence that Tat can inhibit PKR activity by both RNA-dependent and RNA-independent mechanisms. Tat inhibited PKR activation by the non-RNA activator heparin, and also suppressed PKR basal level autophosphorylation in the absence of RNA. However, when Tat and dsRNA were preincubated, the amount of Tat required to inhibit PKR activation by dsRNA depended on the dsRNA concentration. In addition to its function in vitro, Tat can also reverse translation inhibition mediated by PKR in COS cells. The Tat amino acid sequence required for interaction with PKR was mapped to residues 40-58, overlapping the hydrophobic core and basic region of HIV-1 Tat. Alignment of amino acid sequences of Tat and eIF-2alpha indicates similarity between the Tat-PKR binding region and the residues around the eIF-2alpha phosphorylation site, suggesting that Tat and eIF-2alpha may bind to the same site on PKR.
Archives of Biochemistry and Biophysics 02/2000; 373(2):361-7. · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR). We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR. Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA.
The EMBO Journal 12/1999; 18(21):6106-18. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice. All three TR isozymes contain a selenocysteine residue that is located in the penultimate position at the carboxyl terminus and which is encoded by a UGA codon. The generation of reactive oxygen species in a human carcinoma cell line was shown to result in both the oxidation of the selenocysteine in TR1 and a subsequent increase in the expression of this enzyme. These observations identify the carboxyl-terminal selenocysteine of TR1 as a cellular redox sensor and support an essential role for mammalian TR isozymes in redox-regulated cell signaling.
Journal of Biological Chemistry 09/1999; 274(35):24522-30. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A20 is a Cys2/Cys2 zinc finger protein which is induced by a variety of inflammatory stimuli and which has been characterized as an inhibitor of cell death by a yet unknown mechanism. In order to clarify its molecular mechanism of action, we used the yeast two-hybrid system to screen for proteins that interact with A20. A cDNA fragment was isolated which encoded a portion of a novel protein (TXBP151), which was recently found to be a human T-cell leukemia virus type-I (HTLV-I) Tax-binding protein. The full-length 2386 bp TXBP151 mRNA encodes a protein of 86 kDa. Like A20, overexpression of TXBP151 could inhibit apoptosis induced by tumour necrosis factor (TNF) in NIH3T3 cells. Moreover, transfection of antisense TXBP151 partially abolished the anti-apoptotic effect of A20. Furthermore, apoptosis induced by TNF or CD95 (Fas/APO-1) was associated with proteolysis of TXBP151. This degradation could be inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of the cowpox virus-derived inhibitor CrmA, suggesting that TXBP151 is a novel substrate for caspase family members. TXBP151 was indeed found to be specifically cleaved in vitro by members of the caspase-3-like subfamily, viz. caspase-3, caspase-6 and caspase-7. Thus TXBP151 appears to be a novel A20-binding protein which might mediate the anti-apoptotic activity of A20, and which can be processed by specific caspases.