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At least 19 recordsLinked to original sources
PubMed1992

Alterations in tumor angiogenesis associated with stable expression of the HIV tat gene.

Recent evidence suggests that the human immunodeficiency virus type 1 (HIV) trans-activator gene (tat) has transforming properties and may be a causative factor in the development of certain types of cancers, in particular Kaposi's sarcoma (i.e., Vogel J. et al. Nature 335:606-611, 1988). To help elucidate the potential role or roles of the HIV tat gene in neoplastic transformation, cell lines were constructed that constitutively express a functional tat gene product. HeLa cells were coelectroporated with two plasmids, one containing the HIV tat gene in an expression cassette and another containing the dominant selectable marker gene xanthine guanine phosphoribosyltransferase (XGPRT). After XGPRT selection, single-cell clones that expressed a functional tat protein were identified by measuring chloramphenicol acetyltransferase (CAT) activity after electroporating a plasmid containing the CAT gene transcriptionally controlled by HIV trans-activation-responsive region (tar). Phenotypic alterations resulting from the expression of tat were then determined. Control cells and tat-expressing cells grew at similar rates in culture. However, when grown as tumors in nude mice, tat-expressing cells produced a lower percentage of tumors, and the tumors that were produced either regressed, stopped growing, or grew at a very reduced rate compared with cells not expressing tat. These differences may have resulted from a tat-associated reduction in neovascularization in the tumors. A comparison of total cellular proteins by two-dimensional polyacrylamide gel electrophoresis indicated only one reproducible alteration in a polypeptide of approximately 44 kDa and pl of approximately 6.2 associated with tat expression. These cells may be very useful in future in vitro and in vivo studies designed to examine the effects of HIV tat on endothelial and vascular smooth-muscle cells and the role of tat in the etiology of Kaposi's sarcoma.

Animals↗
PubMed1996

Transcriptional up-regulation of interleukin 4 receptors by human immunodeficiency virus type 1 tat gene.

The human immunodeficiency virus type 1 (HIV-1) regulatory gene, tat, encodes an early transactivator protein (Tat) necessary for virus replication. We have reported that the HIV-1 tat gene can up-regulate interleukin 4 receptors (IL-4R) however, the mechanism of this up-regulation is not understood. We now show that in Raji cells, 125I-labeled IL-4 cross-linked to three proteins of 140, 70, and 63 kDa, which were immunoprecipitated with an antibody to the human IL-4R. Although this level of all three IL-4 binding proteins increased in tat-transfected cells, the binding characteristics of IL-4R on control or mock transfected control and tat-transfected cells remained similar. The exogenous recombinant Tat protein or supernatant of tat transfected Raji cells also up-regulated the expression of the IL-4R on two renal cell carcinoma cell lines in a concentration-dependent manner. The actinomycin D chase experiments revealed that the half-lives of the IL-4R protein (t1/2 3.5 hr) and mRNA transcripts (t1/2 2.5 hr) were similar in both control and tat-transfected cells. In contrast, nuclear run-on experiments revealed that the rate of the IL-4R mRNA transcription increased 3- to 5-fold in Raji-tat compared to Raji cells. These data indicate that the HIV-1 tat gene up-regulates IL-4R expression by increasing the transcription rate rather than posttranscriptional stabilization of either the mRNA or the protein. HIV-tat inducible exogenous tumor necrosis factor (TNF-alpha) did not up-regulate IL-4R and IL-4R inducible activation of signal transducers and activators of transcription (STAT-6) was not observed by Tat even though IL-4R were up-regulated. These results allow us to speculate that HIV-1 tat may interact directly with the IL-4R gene and up-regulate IL-4R transcription.

Antigens, CD↗
PubMed1992

Human immunodeficiency virus type 1 tat gene up-regulates interleukin 4 receptors on a human B-lymphoblastoid cell line.

The human immunodeficiency virus type I (HIV-1) regulatory gene, tat III, is a powerful trans-activator of gene expression from the viral long terminal repeat and is essential for HIV replication. In addition, tat III protein has been shown to be immunosuppressive as indicated by the inhibition of antigen mediated T-cell proliferation. To further test whether tat III might play a direct role in the immunosuppressive effects of HIV-1 in addition to its role in virus replication, we examined the regulation of interleukin 4 (IL-4) receptors on a human B-lymphoblastoid cell line (Raji) transfected with HIV-1 tat gene (Raji-tat III). We used radioligand receptor binding analysis for cell surface expression and Northern blot analysis for the expression of human IL-4 receptor gene in Raji-tat III cells. Control Raji cells expressed 1383 +/- 361 (SE; n = 3) IL-4 binding sites/cell with a dissociation constant (Kd) of 144 +/- 27 pM (n = 3). However, Raji-tat III cells expressed about three times higher IL-4 receptors (4000 +/- 633 IL-4 binding sites/cell; P less than 0.03 compared to Raji cells) with a similar Kd of 273 +/- 90 pM (n = 3; P greater than 0.05 compared to Raji cells). Whereas both Raji and Raji-tat III cells exhibited a single mRNA species (approximately 4 kilobases) of IL-4 receptors by Northern blot analysis, the mRNA level was about 3-fold higher in Raji-tat III cells compared to Raji cells. Cycloheximide inhibited the expression of IL-4 receptors by 50% in about 2 h in both cell types indicating both the half-life of IL-4 receptors and the requirement for protein synthesis for the tat III up-regulation of IL-4 receptors. Since IL-4 under certain circumstances has been shown to be immunosuppressant, our observation that the HIV-1 tat gene up-regulates IL-4 receptors suggests the possibility that the immunosuppressive effects of HIV-1 are mediated at least in part through IL-4 receptors.

Cell Line↗
PubMed1997

Bovine immunodeficiency virus tat gene: cloning of two distinct cDNAs and identification, characterization, and immunolocalization of the tat gene products.

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.

3T3 Cells↗
PubMed1992

Identification and characterization of the bovine immunodeficiency-like virus tat gene.

A cDNA clone of the bovine immunodeficiency-like virus (BIV) trans-activator gene (tat) was identified and characterized. The tat cDNA clone was generated by splicing, and on the basis of sequence analysis, the Tat protein was found to be encoded entirely by the first exon. It is 103 amino acids in size and shares sequence homology with the human immunodeficiency virus (HIV) Tat. The BIV tat clone can trans activate the BIV promoter effectively, as measured by the expression of the bacterial chloramphenicol acetyltransferase gene, when transfected into bovine cells. Besides activating the BIV promoter, the BIV Tat can also trans activate the HIV promoter effectively. It is possible that BIV Tat and HIV Tat employ similar mechanisms in trans activation of the viral long terminal repeat-directed gene expression.

Amino Acid Sequence↗
PubMed1988

Transcriptional but not translational regulation of HIV-1 by the tat gene product.

Human immunodeficiency virus-1 (HIV-1), which causes AIDS (acquired immune deficiency syndrome), possesses an essential gene, tat, whose product, acting through the long terminal repeat (LTR) sequences of HIV-1, activates viral genes and replication. The mechanism by which tat trans-activates HIV genes is unclear. Some studies have reported that an increase in messenger RNA accumulation directed by the HIV-1 LTR can explain the action of tat, but others suggest that this increase in mRNA levels can only partially explain trans-activation, and that translational control mechanisms may also be involved. To test those possibilities we have established an efficient adenovirus system for delivering the HIV-1 LTR attached to a reporter gene (chloramphenicol acetyltransferase; CAT) into cells and monitoring its activity. The HIV-1 LTR expressed from this adenovirus responds to trans-activation in a HeLa cell line constitutively expressing the tat protein by increasing the transcription rate of the HIV-1 LTR and the accumulation of mRNA encoding CAT. In this system the translational efficiency of this CAT mRNA in the cell is unaffected by the presence of tat.

Adenoviridae↗
PubMed1988

Gene transactivation mediated by the TAT gene of human immunodeficiency virus in transgenic mice.

Transgenic mice were generated carrying either the long terminal repeat of Human Immunodeficiency Virus fused to the bacterial chloramphenicol acetyl transferase reporter gene or a control element of the murine alpha A crystallin gene fused to the tat gene of human immunodeficiency virus. By crossing these two strains, progeny were obtained which carried both transgenes. The bacterial reporter gene was specifically transactivated in the eyes of these animals.

Acetyltransferases↗
PubMedDate not supplied

New human and simian HIV-related retroviruses possess functional transactivator (tat) gene.

New human retroviruses antigenically related to HIV and even more closely to STLV-III have been recently isolated from individuals from some West African countries. One of these viruses, HTLV-IVP, was reportedly isolated from lymphocytes of a healthy female prostitute. Another isolate, LAV-2FG, was obtained from an AIDS patient and third, SBL-6669, from an individual with lymphadenopathy. Current epidemiological studies indicate that some of these virus isolates cause immune deficiency whereas others may not or may be less efficient at inducing immune deficiency. Similarly, STLV-III apparently does not cause immune deficiency in its natural host, African green monkey. A novel feature of HIV is the possession of a gene termed tat, which is implicated in its pathobiology. We report here that, like HIV, HTLV-IVP, LAV-2FG (HIV-2) and SBL-6669, as well as STLV-IIIAGM possess the putative tat gene, irrespective of their pathogenic potential in vivo. Interestingly, HTLV-IVP/LAV-2FG long terminal repeat (LTR) is equally well transactivated by the HTLV-IVP/LAV-2FG and HTLV-IIIB tat function, HTLV-IIIB LTR responds better to its own tat function.

Acquired Immunodeficiency Syndrome↗
PubMed2005

[The fusion construction of HIV-1 Tat gene and efficient expression in E.coli].

AIM: To express high-level the Tat protein in E.coli. METHODS: Full-length HIV-1 Tat gene was amplified artificially by PCR and Tat gene was mutated site-specifically (substitution the codons AAG encoding the lysine at the 28th and the 50th site by the CAG encoding glutamine) in order to eliminate the transcriptional activity of Tat protein. The site-mutated Tat gene was fused with chaperone10 gene, and then was subcloned into vector pET28a. The recombinant plasmid was expressed in E.coli BL21(DE3). The expressed products were identified by Western blot. RESULTS: Full-length HIV-1 Tat gene was amplified successfully by three rounds of PCR. The recombinant plasmid pET28a-chaperone 10-Tat was expressed efficiently in E.coli BL21(DE3). Western blot analysis showed the expressed Tat fusion protein with relative molecular mass (M(r)) 24 000 could bind to anti-His-tag monoclonal antibody. CONCLUSION: Full-length HIV-1 Tat gene was cloned and chaperone 10-Tat fusion protein was expressed efficiently in E.coli BL21(DE3), which will lay the foundation for researching the pathogenic effect of HIV-1 Tat on AIDS.

Animals↗
PubMed2000

[Effects of human immunodeficiency virus type 1 nef and tat genes on rat PC12 pheochromocytoma cells].

The regulatory genes nef and tat of the human immunodeficiency virus type 1 (HIV-1) were transferred into the rat pheochromocytoma cells (line PC12) under the control of the eukaryotic promoters. Proliferative activity of the PC12 cells transfected with the tat HIV-1 gene was substantially increased as compared to the control. Conversely, the nef gene introduced into the cultivated PC12 cell caused inhibition of their proliferative activity and formation of cell agglomerates resembling in morphology the multinuclear syncytial cells. Thus, our results suggest that the tat gene activates proliferation of the cultivated PC12 cells, whereas the nef gene inhibits proliferation of the same cells. We have obtained for the first time a direct indication for the possible role of the nef gene in formation of multinuclear T-lymphocyte and macrophage syncytium in HIV-1-infected patients. The HIV-1 nef and tat genes had no significant effect on the neuronal differentiation of the PC12 cells induced by the nerve growth factor (NGF).

Animals↗
PubMed1993

Extravasation and transcytosis of liposomes in Kaposi's sarcoma-like dermal lesions of transgenic mice bearing the HIV tat gene.

Transgenic mice bearing the HIV tat gene develop dermal lesions resembling a common malignant tumor in AIDS, Kaposi's sarcoma (KS). To evaluate the permeability characteristics of these lesions and the therapeutic potential of drug-carrying liposomes, we have studied the localization of sterically stabilized liposomes, which show long circulation time in blood and increased accumulation in tumors. Liposomes encapsulating colloidal gold were injected intravenously into transgenic mice bearing KS lesions, and tissues were processed 24 hours later for both electron microscopy and for light microscopy with silver enhancement. Liposomes and silver marker were detected predominantly in the dermis surrounding the early and mature KS lesions, which were characterized by a proliferation of fibroblast-like spindle cells and abnormal blood vessels close to the epidermis. The silver-enhanced gold marker often surrounded vascular channels and scattered erythrocytes. As determined by electron microscopy, some spindle cells and macrophages had ingested intact liposomes. Transendothelial transport of liposomes was observed both through open channels between endothelial cells and also through endothelial cells lining intact vessels. Both extravasation and transcytosis of liposomes through irregular endothelium were much higher in KS lesions than in the adjacent normal skin. The high accumulation of sterically stabilized liposomes in KS lesions and their intracellular uptake by some spindle cells enhances their potential as carriers of chemotherapeutic agents against this neoplasm.

Animals↗
PubMed1999

[Sequence analysis of human immunodeficiency virus type 1 tat gene among the long-term HIV infected non-progressoris in Yunnan province].

OBJECTIVE: HIV-1 tat gene is one of its regulatory genes necessary for its replication. This study is to explore whether the variation of tat gene influences the AIDS progress of HIV infected individual. METHODS: We carried out investigation on tat gene of HIV among the long term HIV infected non-progressors and the commonly infected individuals. Mononuclear cells of peripheral blood were isolated and cellular DNA was extracted. Nested PCR method was used to amplify tat gene and their sequences were analyzed. RESULTS: Blood were collected from 22 HIV infected individuals, of which 11 cases were infected with B subtype strains based on previous env gene analysis and belonged to long term non-progressors according to clinical features and immunology. The other 11 cases were common HIV infection of which 5 subtype B and 6 subtype C were found. Both long term non-progressor group (subtype B) and the commonly HIV infectedly group (subtype B) had constantly four amino acids different from that of international B subtype consensus sequence The both groups clustered randomly on the phylogenetic tree. In commonly HIV infected subtype C group there were 6 amino acids different from that of international subtype C. The variation inside the tatgene of common subtype C group was small. CONCLUSIONS: There was no regular variation and significant difference between long term HIV infected non-progressive group and commonly HIV infected subtype B group. There is no evidence showing that progressive speed of AIDS correlated with the tat sequence profile of HIV-1.

Amino Acid Sequence↗
PubMed1990

Insertional gene synthesis, a novel method of assembling consecutive DNA sequences within specific sites in plasmids. Construction of the HIV-1 tat gene.

The construction of the HIV-1 tat gene using a novel method termed insertional gene synthesis (IGS) is described. IGS is used to assemble a gene or any DNA sequence in a stepwise manner within a plasmid containing a single stranded DNA phage origin of replication. The IGS method is based upon consecutive targeted insertions of long DNA oligonucleotides (greater than 100 bases) within the plasmid by oligonucleotide-directed mutagenesis. IGS therefore involves synthesis of only a few oligonucleotides corresponding to one strand of a gene. Furthermore, the gene is synthesized directly adjacent to bacterial gene regulatory sequences for direct expression. Using this approach, the 261 bp tat gene was assembled in three successive cycles adjacent to the lac promoter in the pEMBL-derivative, pKH125. The 15 kD tat protein was produced from this synthetic gene in E. coli upon IPTG induction. However, it was necessary to tightly control the expression of tat by including the lac I gene directly within the tat expression vector.

Amino Acid Sequence↗
PubMed1990

Control of the interferon-induced 68-kilodalton protein kinase by the HIV-1 tat gene product.

The tat-responsive region (TAR) of the human immunodeficiency virus-1 (HIV-1) exhibits a trans-inhibitory effect on translation in vitro by activating the interferon-induced 68-kilodalton protein kinase (p68 kinase). Productive infection by HIV-1 was shown to result in a significant decrease in the amount of cellular p68 kinase. The steady-state amount of p68 kinase was also reduced in interferon-treated HeLa cell lines stably expressing tat, as compared to the amount of the kinase in interferon-treated control HeLa cells. Thus, the potential translational inhibitory effects of the TAR RNA region mediated by activation of p68 kinase may be downregulated by tat during productive HIV-1 infection.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

Modulation of TAT gene induction by glucocorticoids involves a neutralizing sequence.

Recent studies have indicated that two elements in addition to the glucocorticoid response element (GRE) are involved in the induction of the endogenous TAT gene in FuS-5 rat hepatoma cells. The first is the 21 bp glucocorticoid modulatory element (GME) at -3648 bp, which causes reporter constructs to display both a left shift in the dose-response curve for glucocorticoids and increased percentages of agonist activity for antiglucocorticoids. The second is a negative element at -3340 to -3050 that blocks the action of the GME. This last observation raised the question of how GME activity can be expressed in Fu5-5 cells in the intact TAT gene that contains both the GME and the negative element. The present study identifies a third element, a "neutralizing" sequence, that restores the activity of the GME even when otherwise inactivated by the negative element. This neutralizing sequence was located within the region surrounding the GREs of the TAT gene but is separate from the GREs. The activity of the individual GME and negative elements was found to depend upon spacing. However, in combination with the natural GRE, the native TAT gene spacing of the GME and negative elements was able to reproduce the activity of the intact gene. Thus, a total of three additional elements (an activator, a negative element, and a neutralizer) appear to cooperate with the GREs in glucocorticoid induction of the TAT gene in Fu5-5 cells. While such a grouping of elements may be novel among steroid regulated genes, it is a not uncommon occurrence for the transcriptional control of other genes.

Animals↗