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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied regulation of human immunodeficiency virus-1 (HIV-1) transcription by Tat and, for comparative purposes, by the adenovirus E1A protein. These two trans-activators exerted different effects. Two classes of HIV-1-promoted cytoplasmic RNA were detected, one class corresponding to full-length transcripts and the other to transcripts ending 55 and 59 nucleotides from the transcription start. Tat increased the level of the full-length class only, whereas E1A increased the levels of both classes of RNA. We also measured the effects of Tat and E1A on RNA synthesis rates. Without trans-activators, HIV-1-directed transcription was relatively weak and exhibited a marked polarity. Both Tat and E1A dramatically increased promoter-proximal transcription, while only Tat suppressed transcriptional polarity. Mutations in the TAR element did not influence basal transcription rates or the response to E1A, but eliminated trans-activation by Tat. We propose that Tat acts through TAR to increase initiation complex formation on the HIV-1 promoter and to stabilize complexes during elongation.
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PMID:HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation. 255 66

Five regions of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) have been shown to be important in the transcriptional regulation of HIV in HeLa cells. These include the negative regulatory, enhancer, SP1, TATA, and TAR regions. Previous studies in which purified SP1 was used showed that the three SP1-binding sites in the HIV LTR were important in the in vitro transcription of this promoter. However, no studies to ascertain the role of each of these SP1-binding sites in basal and tat-induced transcriptional activation in vivo have been reported. To determine the role of SP1 sites in transcriptional regulation of the HIV LTR in vivo, these sites were subjected to oligonucleotide mutagenesis both individually and in groups. The constructs were tested by DNase I footprinting with both oligonucleotide affinity column-purified SP1 and partially purified HeLa extract and by chloramphenicol acetyltransferase assays in both the presence and absence of the tat gene. Mutagenesis of each SP1-binding site resulted in minimal changes in basal and tat-induced transcriptional activation. Mutations involving alterations of SP1 sites I and II, I and III, or II and III also resulted in minimal decreases in basal and tat-induced transcriptional activation. However, mutagenesis of all three SP1-binding sites resulted in a marked decrease in tat induction. The latter mutation also greatly decreased DNase I protection over the enhancer, TATA, and TAR regions when partially purified HeLa nuclear extract was used. Mutagenesis of the HIV LTR SP1 sites which converted them to consensus high-affinity SP1-binding sites with the sequence GGGGCGGGGC resulted in increased tat-induced gene expression compared with the wild-type HIV LTR template. These results suggest that SP1, through its interaction with other DNA-binding proteins, is critical for in vivo transcriptional regulation of HIV.
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PMID:Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat. 265

Transcription of the human immunodeficiency virus type 1 (HIV-1) is regulated by viral proteins and cellular factors that bind to the viral long terminal repeat (LTR). At least five regions of the HIV LTR serve as binding sites for HeLa cellular proteins. One region containing two copies of the sequence GGGACTTTCC functions as an enhancer element for HIV transcriptional regulation. Another region between -17 and +44 known as the TAR region contains two copies of the sequence CTCTCTGG and is also important in tat-induced activation of the HIV LTR. HeLa cell extracts were used to purify cellular proteins binding to portions of the enhancer region (EBP-1) and the TAR region (UBP-1) by a combination of conventional and DNA affinity chromatography. Several species of proteins of between 55 and 60 kd were found to bind to specific sequences in the enhancer region and these proteins also bound to a portion of the NF-kappa B binding site in the immunoglobulin kappa enhancer. Two proteins of between 61 and 63 kd were the major species found to bind to specific sequences in the TAR region and fractions containing these proteins also bind to the TATA region. Both UBP-1 and EBP-1 exhibited specific binding as demonstrated by both UV cross-linking and DNase I footprinting. Mutations of either the enhancer or TAR regulatory regions prevented binding of these purified factors. These results demonstrate the binding of highly purified cellular proteins to important transcriptional regulatory regions in the HIV LTR.
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PMID:Purification of the human immunodeficiency virus type 1 enhancer and TAR binding proteins EBP-1 and UBP-1. 313 13

The synthesis of a gene for the HIV TAT protein is described using a novel approach that capitalises on the ability to synthesise oligonucleotides of greater than 100 bp in length. It involves the synthesis of large oligomers covering one strand of the desired gene in its entirety and the use of small complementary bridging and adapter oligonucleotides to direct the assembly and cloning of the large oligomers. After ligation to the cloning vector the partially single stranded intermediate is transformed directly into the recipient bacterial host where the plasmid is repaired. The synthetic tat gene has been expressed in HeLa cells and is shown to trans-activate TAR+ but not TAR- HIV LTR-CAT constructs.
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PMID:Synthesis of a gene for the HIV transactivator protein TAT by a novel single stranded approach involving in vivo gap repair. 328 69

In this study we have defined the in vitro requirements for transcriptional regulation of the HIV-2 LTR in response to the HIV-1 and HIV-2 Tat proteins and addressed potential mechanisms of Tat function. HIV-2 contains a duplicated TAR RNA stem-loop structure in contrast to the single stem-loop structure found in HIV-1 TAR RNA. We demonstrated that the HIV-2 proximal TAR RNA stem-loop structure was more important for in vitro transcriptional activation by the HIV-1 and HIV-2 Tat proteins than the distal TAR RNA stem-loop though this downstream TAR element itself was able to confer Tat-responsiveness. The role of the two HIV-2 TAR RNA stem-loop bulge sequences was less critical than the loop sequences for in vitro transcriptional activation by Tat. In addition, we demonstrated that replacing the HIV-2 TATA element with that of HIV-1 markedly reduced the overall level of Tat activation. The role of the Tat-1 and Tat-2 proteins on the synthesis of HIV-1 and HIV-2 promoter proximal and promoter distal transcripts was then investigated. In contrast to the HIV-1 promoter, the HIV-2 promoter generated abundant levels of short transcripts in vitro transcription assays likely due to the structure of its duplicated TAR element. Both Tat-1 and Tat-2 increased the level of transcripts extending to the end of the HIV-1 and HIV-2 TAR elements as well as the level of transcripts extending more than 500 nucleotides from the transcription initiation site. However, the synthesis of transcripts within 30 nucleotides of the HIV-2 LTR transcription initiation site was unchanged in either the presence or absence of Tat while the level of transcripts extending increasing distances from the HIV-2 LTR transcription initiation site were progressively stimulated in the presence of Tat. Though the HIV-1 Tat protein was a stronger inducer of HIV-1 LTR transcription than the HIV-2 Tat protein, we did not detect differences in the binding of these proteins to the HIV-1 and HIV-2 TAR RNAs. This suggested that differences in their transactivation properties may be due to alterations in their association with RNA polymerase II or associated elongation factors. (ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tat functions to stimulate the elongation properties of transcription complexes paused by the duplicated TAR RNA element of human immunodeficiency virus 2. 749 Jul 54

The TAR sequence at the 5'-termini of all HIV-1 mRNA species forms a stable structure that is responsible for both transcriptional and translational regulation of HIV-1. Previously we and others reported that purified TAR RNA synthesized by in vitro transcription could activate two interferon-induced enzymes, the protein kinase (PKR) and 2-5A-synthetase. Because the PKR- and 2-5A-systems block protein synthesis initiation and induce RNA decay, respectively, these findings suggested mechanisms for the control of HIV-1 replication by the interferon system. To determine if contaminating dsRNA from in vitro transcription reactions was responsible for this effect, as suggested by Gunnery et al. 1990, (Proc., Natl. Acad. Sci. USA 87, 8687), we have reexamined these findings using chemically synthesized TAR (nucleotides +1 to +57). TAR RNA is shown here to have an intrinsic ability to activate PKR and 2-5A-synthetase. In contrast, a mutant form of TAR designed to have a disrupted secondary structure did not stimulate either enzyme. Chemically synthesized TAR mimicked other dsRNA species in its ability to activate and inhibit PKR at low and high RNA concentrations, respectively. HIV-1 TAT protein inhibited activation of PKR by HIV-1 TAR RNA suggesting an escape mechanism for the virus.
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PMID:HIV-1 TAR RNA has an intrinsic ability to activate interferon-inducible enzymes. 752 41

HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR. TAR is located at the immediate 5' end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process.
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PMID:Requirement for HIV-1 TAR sequences for Tat activation in rodent cells. 753 Mar 99

The course of drug development for the treatment of HIV-1 infection and AIDS is being revolutionized by high-resolution structures of essential viral proteins. We survey the impact on drug design of the recently elucidated structural knowledge of two essential enzymes, reverse transcriptase and protease, and three new targets, the viral integrase and the gene regulatory protein-RNA interactions, Tat-TAR and Rev-RRE.
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PMID:Progress in anti-HIV structure-based drug design. 754 68

We have constructed a new retroviral vector by making modifications to the commonly used Moloney murine leukemia virus (MoMLV) based vector in the long terminal repeat (LTR). The changes include replacement of a portion of the U3 region of the MoMLV LTR with a hybrid regulatory element consisting of the human cytomegalovirus immediate-early enhancer/promoter (CMV-IE) together with the human immunodeficiency virus transactivation response element (HIV-TAR). Transfection of chloramphenicol acetyl transferase (CAT) reporter constructs into a variety of human cell lines showed that the hybrid LTR with the CMV-IE/HIV-TAR enhancer/promoter exhibited basal expression levels which were 10- to 50-fold higher than those obtained from the wild-type MoMLV-LTR enhancer/promoter. Expression from the recombinant LTR was further increased in the presence of the HIV-Tat protein, and surprisingly, Tat up-regulated transcription from both the HIV and the MoMLV TATA boxes. In contrast, a MoMLV enhancer/promoter containing only the HIV-TAR element in the LTR did not respond to Tat. When stably transfected into an amphotropic packaging cell line, the modified retroviral vector containing the hybrid LTR plus an extended packaging signal consistently gave higher titres of retrovirus than did the parental MoMLV based vector. Higher basal expression levels which can be further upregulated by Tat, together with more efficient virion production, suggests that the modified vector should be superior for anti-HIV gene therapy applications as well as for other more general applications in human gene therapy.
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PMID:Retroviral vector with a CMV-IE/HIV-TAR hybrid LTR gives high basal expression levels and is up-regulated by HIV-1 Tat. 755 87

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.
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PMID:Molecular cloning and characterization of a TAR-binding nuclear factor from T cells. 757 25


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