Transcriptional activation of homologous viral long terminal repeats by the human immunodeficiency virus type 1 or the human T-cell leukemia virus type I tat proteins occurs in the absence of de novo protein synthesis (original) (raw)
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Analysis of Tat transactivation of human immunodeficiency virus transcription in vitro
Gene expression, 1992
The HIV Tat protein is a potent transactivator of HIV transcription, increasing both RNA initiation and elongation. We now demonstrate that purified, full-length 86 amino acid Tat protein specifically transactivates the HIV LTR in vitro to a high level (25- to 60-fold). Tat transactivation was specifically blocked by anti-Tat serum, but not preimmune serum. Tat did not transactivate transcription from the control adenovirus major late promoter (AdMLP). HIV transcription was blocked at various functional steps during initiation and elongation complex formation. Similar to the control AdMLP, HIV basal initiation complex assembly was sensitive to the addition of 0.015% sarkosyl prior to the addition of nucleoside triphosphates. Resistance to 0.05% sarkosyl required the addition of G, C, and U, which constitute the first 13 bases of the HIV RNA transcript. The addition of Tat to the in vitro transcription relieved the 0.015% sarkosyl block. These Tat-induced complexes were sensitive to ...
Journal of Molecular Biology, 1997
We investigated the role of a 54-nucleotide region (200 to 253) located downstream of the HIV-1 long terminal repeat (LTR) on virus gene expression and found, using RT-PCR and p24 CA analysis, that deletion of this region inhibited synthesis of both viral RNA and protein. CAT assays showed that these results were attributable to decreased transcription ef®ciency of the HIV-1 LTR and not to the stability of the RNA transcripts produced. Further deletional analysis and transfection studies showed that the most important sequences with regard to proviral DNA expression were located between nucleotide positions 218 and 237. Furthermore, substitutional mutational analysis showed that a CTCTCTC sequence at positions 227 to 233, homologous to the pyrimidine-rich initiator (Inr) region found in several promoters, was required for ef®cient production of both viral RNA and protein. Deletion of the sequence 200 to 217, homologous to the interferon-stimulated response element (ISRE), resulted in impaired LTR promoter activity and decreased synthesis of viral RNA and protein. However, when the latter region was replaced by homologous ISRE sequences from an interferon-stimulated gene (ISG-54), an even more severe effect on HIV gene expression and replication was observed, suggesting that ISRE-like sequences in HIV act differently from homologous sequences in interferon-responsive cellular genes.
Virology, 1996
The HIV-1 Tat protein activates transcription of the viral LTR promoter through interaction with the nuclear transcription machinery of the host cell. Tat can also activate the LTR promoter in a paracrine or inter-cellular manner by a yet unknown mechanism. One possibility is that Tat protein itself is secreted by cells and taken up by other cells. According to this mechanism, inter-cellular transcriptional activation by Tat should be very similar to intra-cellular trans-activation in Tatproducing cells. A large number of cytokine genes was recently reported to be Tat-responsive, raising the possibility that such cytokines and the corresponding cellular transduction pathways are involved in inter-cellular Tat action. The transcriptional events in such an indirect route are likely to differ from intra-cellular Tat action. To discriminate between a direct or indirect mechanism of inter-cellular Tat action, we compared the activity of a set of Tat mutants and different promoter constructs in inter-cellular and intra-cellular transcriptional activation. Identical results were obtained in both assays, suggesting that Tat protein itself is exported by one and transported into the nucleus of another cell. The demonstration that Tat antibodies specifically inhibit the inter-cellular route is also consistent with cell-to-cell transport of the Tat protein. Furthermore, we found that the second Tat coding exon, including the RGD motif that has been proposed to interact with an integrin receptor, is not required for cellular uptake of the Tat protein.
Journal of Virology, 2000
The current human immunodeficiency virus type 1 (HIV-1) shows an increasing number of distinct viral subtypes, as well as viruses that are recombinants of at least two subtypes. Although no biological differences have been described so far for viruses that belong to different subtypes, there is considerable sequence variation between the different HIV-1 subtypes. The HIV-1 long terminal repeat (LTR) encodes the transcriptional promoter, and the LTR of subtypes A through G was cloned and analyzed to test if there are subtype-specific differences in gene expression. Sequence analysis demonstrated a unique LTR enhancer-promoter configuration for each subtype. Transcription assays with luciferase reporter constructs showed that all subtype LTRs are functional promoters with a low basal transcriptional activity and a high activity in the presence of the viral Tat transcriptional activator protein. All subtype LTRs responded equally well to the Tat trans activator protein of subtype B. This result suggests that there are no major differences in the mechanism of Tat-mediated trans activation among the subtypes. Nevertheless, subtype-specific differences in the activity of the basal LTR promoter were measured in different cell types. Furthermore, we measured a differential response to tumor necrosis factor alpha treatment, and the induction level correlated with the number of NF-B sites in the respective LTRs, which varies from one (subtype E) to three (subtype C). In general, subtype E was found to encode the most potent LTR, and we therefore inserted the core promoter elements of subtype E in the infectious molecular clone of the LAI isolate (subtype B). This recombinant LAI-E virus exhibited a profound replication advantage compared with the original LAI virus in the SupT1 T-cell line, indicating that subtle differences in LTR promoter activity can have a significant impact on viral replication kinetics. These results suggest that there may be considerable biological differences among the HIV-1 subtypes.
Cell Cycle-Regulated Transcription by the Human Immunodeficiency Virus Type 1 Tat Transactivator
Journal of Virology, 2000
Cyclin-dependent kinases are required for the Tat-dependent transition from abortive to productive elongation. Further, the human immunodeficiency virus type 1 (HIV-1) Vpr protein prevents proliferation of infected cells by arresting them in the G 2 phase of the cell cycle. These findings suggest that the life cycle of the virus may be integrally related to the cell cycle. We now demonstrate by in vitro transcription analysis that Tat-dependent transcription takes place in a cell cycle-dependent manner. Remarkably, Tat activates gene expression in two distinct stages of the cell cycle. Tat-dependent long terminal repeat activation is observed in G 1 . This activation is TAR dependent and requires a functional Sp1 binding site. A second phase of transactivation by Tat is observed in G 2 and is TAR independent. This later phase of transcription is enhanced by a natural cell cycle blocker of HIV-1, vpr, which arrests infected cells at the G 2 /M boundary. These studies link the HIV-1 Tat protein to cell cycle-specific biological functions.
Journal of virology, 1989
The human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR) is the site of activation of the HIV tat protein. However, additional transactivators, such as the adenovirus E1A and herpesvirus ICPO proteins, have also been shown to be capable of activating the HIV LTR. Analysis of adenovirus mutants indicated that complete transactivation of the HIV LTR was dependent on both the E1A and E1B proteins. To determine which regions of the HIV LTR were important for complete E1A/E1B activation, a variety of oligonucleotide-directed mutations in HIV transcriptional regulatory domains were assayed both in vivo and in vitro. S1 nuclease analysis of RNA prepared after transfection of these HIV constructs into HeLa cells infected with wild-type adenovirus indicated that the enhancer, SP1, TATA, and a portion of the transactivation-responsive element were each required for complete E1A/E1B-mediated activation of the HIV LTR. These same promoter elements were required for both basal an...
Molekuliarnaia genetika, mikrobiologiia i virusologiia, 2009
An important problem in the development of gene therapy approaches in oncology is the necessity of using promoters providing specific and high level of gene expression in tumor cells. To solve this problem, we used inducible system of gene expression regulation (Tat-TAR-system), which is utilized by human immunodeficiency virus (HIV). tat and tk-HSV genes, as well as a fragment of LTR HIV-1, were cloned in the retrovirus vector, tk-HSV gene was under control of the LTR HIV-1 fragment. Potential capacity of these constructions for transactivating tk-HSV gene transcription was studied. Basal expression level of this gene was defined in transient transfection of HEK293 cells. It was shown that specific transactivation of the tk-HSV gene was controlled by the LTR HIV-1 fragment in lung carcinoma cells Calu-1, permanently transfected by the tat gene construction. The effect of transactivation of tk-HSV transcription in Tat-TAR-system was demonstrated in Calu-1 cells in conditions of cont...
AIDS, 1997
Objective: To determine whether the HIV-1 transactivator protein Tat acts as a DNA sequence-specific transcription factor and activates transcription from a heterologous TATAA element in the absence of the trans-activation response (TAR) element and other sequences in the HIV-1 long terminal repeat (LTR). Design: Activating protein-1 (AP-1) and Tat-induced transcription were assessed using Jun and hybrid Tat/Jun-expression plasmids and reporter gene constructs which contained AP-1 binding sites upstream of the rat prolactin TATAA element or an HIV-1 LTR construct in which AP-1 binding sites replaced the TAR element. Methods: Tat-induced transcription was determined following transient transfection of colon epithelial cell lines with reporter gene constructs and Tat/Jun-expression plasmids in which Tat was fused to the DNA binding domain of Jun. Activation of prolactin (PL) and LTR reporter genes was assessed by luciferase (LUC) or chloramphenicol acetyltransferase (CAT) activity in cellular extracts. Results: Cotransfection of cells with Tat/Jun and the AP-1 PL LUC or LTR AP-1 CAT reporter plasmid resulted in a marked increase in reporter gene activity which was comparable with that induced by transfection of cells with several different AP-1 expression plasmids (e.g., JunD, JunB, c-Fos), or that elicited by stimulation of the cells transfected with LTR AP-1 CAT plasmids with phorbol ester or tumor necrosis factor-α. Tat-induced transcription was DNA-mediated since both a Jun DNA binding domain fused to Tat as well as AP-1 binding sites within the promoter were required for the induction of CAT expression. Conclusions: Tat-activated transcription can occur strictly through a heterologous TATAA element independent of TAR and Sp1 binding sites or other HIV-1 LTR sequences. Tat appears to increase transcription initiated through the TATAA element by mechanisms similar to that of DNA sequence-specific transcription factors.