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

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) encode related proteins called Tat-1 and Tat-2, respectively, that bind directly to the TAR RNA element contained at the 5' ends of viral transcripts and thereby stimulate transcription through an as yet unidentified mechanism. The determinants in the HIV-1 TAR element (TAR-1) that specify binding by the Tat-1 protein have been extensively characterized, while little is known about determinants in the HIV-2 TAR element (TAR-2) that specify binding by the Tat-2 protein. The HIV-2 TAR RNA element (TAR-2) is known to be composed of two stem-loop structures. A dinucleotide bulge is found in each stem of TAR-2 RNA, analogous to the crucial trinucleotide bulge in the single stem-loop of HIV-1 TAR RNA that is the primary binding determinant for binding by the HIV-1 Tat protein. Our results of a nuclease digestion analysis demonstrated that the 5' proximal bulge in TAR-2 is significantly less sensitive to digestion by single-strand specific nucleases than the 3' distal bulge, suggesting that the 5' bulge may be involved in tertiary interaction with other regions of TAR RNA. Deletion of both bulges reduced binding in vitro by the Tat-2 protein and largely abolished transactivation in vivo by Tat-2. Deletion of either bulge alone simplified the pattern of protein/RNA complexes in a gel shift assay, but did not reduce the overall binding affinity of Tat-2. Deletion of the 5' bulge reduced Tat-2 transactivation in vivo to a level approximately 30% that of wild-type TAR-2, while deletion of the 3' bulge had no measurable effect in vivo. Our results suggest that each dinucleotide bulge specifies a Tat-2 binding site, but in the wild-type TAR-2 element the 3' bulge binding site does not appear to be utilized in vivo.
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PMID:Functional significance of the dinucleotide bulge in stem-loop1 and stem-loop2 of HIV-2 TAR RNA. 800 32

Human immunodeficiency virus type 1 (HIV-1) RNA contains an extended hairpin structure at the 5' end (the TAR element) that is essential for viral replication. The upper part of the stem-loop structure binds the virally encoded transcriptional activator protein Tat and cellular co-factors, but no clear function for the lower stem region has been established. Here, we report that mutant HIV-1 viruses with base substitutions in the lower stem region are dead, most likely at the level of transcription from an integrated provirus. By using large amounts of these mutant DNA constructs for transfections, revertant viruses with a great variety of genetic changes (point mutations, short deletions) could be isolated in prolonged culture experiments that lasted over 6 months. The pattern and evolution of these changes supported the notion that base-pairing of the lower stem region is essential for optimal HIV-1 replication. The functional and genetic plasticities of this RNA domain and the HIV-1 long terminal repeat promoter are discussed.
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PMID:Evolution of a disrupted TAR RNA hairpin structure in the HIV-1 virus. 801 64

Thirteen genetically altered HIV-1 proviruses were created. These various genomes can be segregated into three groups: (i) a set of tat(-) viruses that have a functional HTLV-I Tax inserted in nef; (ii) a set of tat(-) viruses with Gal4 binding sites inserted in U3 and a Gal4-VP16 cDNA inserted in nef; and (iii) a set of tat(+) HIV genomes that are 5' and 3' TAR(-) and are Gal4-binding-site(+) in U3 and Gal4-VP16(+) in nef. We found that viruses in groups (i) and (ii), although tat(-), were fully complemented for viral gene expression based on quantitative measurements of viral protein synthesis and on the visualization by electron microscopy of the proper assembly of morphologically correct virions. Interestingly, group (i) and (ii) virions were defective in a spreading cytopathic infection when assayed in T-lymphocytes. Group (iii) viruses, although capable of producing intact Tat protein, also could not use Tat for transcription/gene expression because of the TAR(-) genotype. However, this class of viral genomes produced viruses that were highly infectious and cytopathic in primary and in continuously propagated T-lymphocytes. These three groups of viruses are all transcriptionally Tat-TAR independent. Their distinct differences in infectivity/cytopathicity provide genetic evidence that Tat provides a transcriptionally independent function in determining infectivity and cytopathicity in the setting of a spreading viral infection. Given that all HIV virions normally contain four intact copies of TAR RNA, our findings suggest a re-examination of whether Tat could be a virion-TAR-associated protein and the possible implications of this for virus infectivity/cytopathicity.
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PMID:Human immunodeficiency viruses regulated by alternative trans-activators: genetic evidence for a novel non-transcriptional function of Tat in virion infectivity. 802 73

RNA-protein complexes isolated following a gel retardation assay can be footprinted within the gel matrix using the chemical nuclease activities of 4,7-dimethyl-, 5,6-dimethyl-, and 3,4,7,8-tetramethyl-1,10-phenanthroline-copper. These complexes are more reactive than 1,10-phenanthroline-copper but share its reaction preference for bulges and loops. The interaction of the coat protein of R-17 with its viral RNA target and tat- and tat-derived peptides with HIV TAR RNA have been studied. In both cases, the RNA sequence opposite a 2-3 nucleotide bulge are protected. Tat-derived peptides inhibit cleavage at sites which intact tat does not protect. These results are consistent with transcription studies which have suggested that truncation of tat increases nonspecific binding.
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PMID:Footprinting RNA-protein complexes following gel retardation assays: application to the R-17-procoat-RNA and tat--TAR interactions. 803 53

The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.
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PMID:Differential growth kinetics are exhibited by human immunodeficiency virus type 1 TAR mutants. 805 69

Previously it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Tat protein mediates induction of the HIV-1 env expression through a TAR-independent manner in heterologous and homologous promoter systems (Kim and Risser, 1993, J. Virol. 67, 239; Kim and Panganiban, 1993, J. Virol. 67, 3739). To further explore the transactivation of HIV-1 env gene, I examined expression of the env, the bacterial CAT, and the firefly luciferase genes from a heterologous promoter, the major immediate-early promoter (MIEP) of murine cytomegalovirus (MCMV). Here we show that Tat augments gene expression from the MCMV MIEP only when linked to the env gene. Surprisingly, in contrast to the expression from an HIV-1 LTR lacking the TAR element, TAR-independent transactivation of env gene expression from MCMV MIEP did not require the full length Tat protein. In addition, deletion of the previously identified cis-acting Tat-responsive element in env did not affect Tat transactivation of the env gene expression. Thus, there are multiple distinct elements that mediate Tat responsiveness in the absence TAR.
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PMID:Requirement of the human immunodeficiency virus type 1 env gene sequence for TAR-independent trans activation by Tat from the major immediate-early promoter of murine cytomegalovirus. 809 34

The 5'-TAR region of HIV-1 mRNA is highly conserved amongst different HIV-1 isolates. We thus investigated the potential for in vivo targeting of the TAR RNA element by a hammerhead ribozyme. The use of the CAT reporter gene linked to the HIV1-LTR, in transient assays, reveals that a hammerhead ribozyme directed towards the first GUC of HIV-1 mRNA can efficiently inhibit CAT protein expression. We show that this inhibition is sequence-specific and probably due to a cleavage activity rather than an antisense effect. We show also that a hammerhead ribozyme that is inactive in vitro is capable of inhibiting CAT protein expression in a cellular environment. These results suggest that the targeting of the HIV-1 LTR by a hammerhead ribozyme constitutes a viable approach for anti-HIV therapy.
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PMID:Ribozyme targeting of HIV-1 LTR. 809 72

The mismatched double-stranded RNA (dsRNA), poly(I).poly(C12U), also termed Ampligen, exhibits a strong antiviral and cytoprotective effect on cells (human T-lymphoblastoid CEM cells and human T-cell line H9) infected with the human immunodeficiency virus type 1 (HIV-1). Untreated H9 cells infected with HIV-1 start to release the virus 3 days post-infection, while in the presence of 40 micrograms/ml (80 micrograms/ml) of poly(I).poly(C12U) the onset of virus production and release is retarded and does not occur before day 5 (day 6). We demonstrate that poly(I).poly(C12U) markedly extends the duration of the transient increase of 2',5'-oligoadenylate (2-5A) synthetase mRNA level and activity preceding virus production after infection of cells with HIV-1. Treatment of HeLa cells with poly(I).poly(C12U) was found to cause a significant increase in total (activated plus latent) 2-5A synthetase activity; no evidence was obtained that the level of latent (nonactivated) 2-5A synthetase is changed in cells treated with dsRNA plus interferon (IFN). Poly(I).poly(C12U) is able to bind and to activate 2-5A synthetase(s) from HeLa cell extracts. Addition of poly(I).poly(C12U) to HeLa cell extracts results in production of longer 2-5A oligomers (> or = 3 adenylate residues), which are better activators of RNase L. Both free and immobilized poly(I).poly(C12U) also bind to the dsRNA-dependent protein kinase (p68 kinase), resulting in autophosphorylation of the enzyme. Activation of the kinase by the free RNA occurs within a limited concentration range (10(-7) to 10(-6) grams/ml). Addition of HIV-1 Tat protein does not affect binding and activation of p68 kinase to poly(I).poly(C12U)-cellulose but strongly reduces the binding of the kinase to immobilized TAR RNA of HIV-1. We conclude that poly(I).poly(C12U) may antagonize Tat-mediated down-regulation of dsRNA-dependent enzymes.
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PMID:Mode of action of the anti-AIDS compound poly(I).poly(C12U) (Ampligen): activator of 2',5'-oligoadenylate synthetase and double-stranded RNA-dependent kinase. 809 1

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription. While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat. This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes. Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes. The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B. The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not. In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain. Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity. The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.
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PMID:Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat. 818 31

The human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) Tat proteins Tat-1 and Tat-2 stimulate transcription of the viral long terminal repeat (LTR) sequences and are required for efficient viral replication. A class of mutant Tat proteins, termed "transdominant mutants," has been described that possesses relatively low transactivation activity, yet is able to inhibit the function of wild-type Tat. These mutant proteins contain a nonfunctional TAR RNA-binding domain but apparently retain a functional activation domain. A potential limitation for therapeutic use of transdominant mutants described to date is their low but significant basal level of transactivation for the HIV-1 or HIV-2 LTRs. In order to make an improved transdominant mutant, we have constructed Tat-2 proteins that contain mutations in four contiguous arginines at residues 81 to 84 in the RNA-binding domain. Using purified proteins and in vitro RNA-binding assays, we verified that these mutant Tat-2 proteins are defective for TAR RNA binding. We also verified that these mutant Tat-2 proteins bind to a cellular protein kinase in vitro that we have previously shown to bind specifically to the Tat-1 and Tat-2 activation domain. Using plasmid cotransfection assays, we compared the phenotypes of these mutant Tat-2 proteins with the most potent Tat-1 transdominant mutant described to date. One Tat-2 mutant, named "R81-84A," was found to be equivalent to the Tat-1 mutant in ability to inhibit wild-type Tat transactivation of HIV-1 and HIV-2 LTRs. Moreover, the R81-84A mutant possessed a significantly lower basal level of transactivation than the Tat-1 mutant. The R81-84A Tat-2 mutant is therefore a promising reagent for future development as an anti-HIV agent. Additionally, our results suggest that wild-type Tat-2 transactivation of the HIV-2 LTR is especially sensitive to inhibition by transdominant mutants.
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PMID:Construction and characterization of a potent HIV-2 Tat transdominant mutant protein. 820 44


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