UMLS:C0019693 - FACTA Search

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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 type 2 (HIV-2) gene expression is downmodulated by sequence elements downstream of the transcriptional initiation site, corresponding to the U5 region of the long terminal repeat (LTR) and further downstream. This repression appeared to be related more to the length of the sequence intervening the transcriptional initiation site and the coding region than to a particular sequence content. The repressive effect of the downstream segment was not affected by HIV-2 and HIV-1 TAT or by the cytomegalovirus transactivator IE-2 gene. Nor was it affected by T-cell activation signals or by such cytokines as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interferon-gamma (IFN gamma), and interferon-alpha (IFN alpha). In contrast to HIV-1, HIV-2 LTR-directed gene expression was not modulated by TNF-alpha. A specific sequence element, located downstream of the TAR element in the R region, seemed to participate in modulation of gene expression. This element interacted with a nuclear protein with a mobility of about 26 kD. The repressive effect of the downstream sequence was to a certain extent cell type dependent, suggesting the involvement of cell type-specific factors. It was more effective in human lymphocytic CEM cells than in Jurkat cells. This may be relevant to the HIV-2 cell tropism (replication), latency, and virulence.
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PMID:Human immunodeficiency virus type 2 (HIV-2) gene expression: downmodulation by sequence elements downstream of the transcriptional initiation site. 181 41

Substantial evidence indicates that HIV-1 trans-activation by tat protein is mediated through the TAR RNA element. This RNA forms a stem-loop structure containing a three-nucleotide bulge and a six-nucleotide loop. Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site. However, there appears to be no sequence-specific contribution of the six-base loop. We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR. The results indicate that tat interacts with both the bulge and loop regions of TAR. Treatment of TAR RNA with RNase A results in cleavage at U23 and U31, located in the bulge and loop regions, respectively. High concentrations (approximately 2 microM) of Escherichia coli derived tat protein, prepared by standard procedures, gave complete protection of TAR RNA from RNase A cleavage. However, under these conditions, truncated TAR derivatives in which no stem-loop structure is expected to form were also protected, indicating nonspecific binding. In order to obtain a tat preparation with enhanced specificity toward TAR RNA, methods were developed for refolding the recombinant protein. This treatment enhanced the affinity of tat for TAR by approximately 30-fold [Kd(apparent) less than 25 nM] and markedly increased its specificity for the TAR. Again, tat protected TAR RNA from RNase A cleavage at both U23 and U31. Protection was also observed with RNase T1 which cleaves TAR RNA at three G residues in the six-base loop.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage. 186 81

Human immunodeficiency virus type 1 (HIV-1) is viable and mitogen inducible in the absence of its binding sites for the inducible transcription factor NF-kappa B. We have investigated alternative mechanisms for induction of HIV-1 transcription. Using transient transfection assays, we found that transcription from an HIV-1 LTR containing mutant kappa B sites was activated 10- to 20-fold in a variety of human cell types by the phorbol ester phorbol myristate acetate (PMA). The promoter elements conferring this inducibility were localized to the region downstream of nucleotide -70, which contains the TATA and TAR elements and binding sites for transcription factors Sp1 and LBP-1. Synthetic promoters containing only Sp1 sites and a TATA element were also induced in transfection experiments as well as in in vitro transcription experiments with T-cell nuclear extracts. Moreover, promoters containing a TATA box in the absence of Sp1 sites or Sp1 sites in the absence of a TATA box were equally inducible in vitro, as was an RNA polymerase III promoter. The activities of RNA polymerases II and III and of the 38-kDa TATA-binding protein transcription factor IID (TFIID), were not induced by PMA, but electrophoretic mobility shift assays revealed a highly inducible protein-DNA complex that interacted specifically with the TATA sequence. This protein-DNA complex appeared to be much larger than that found with the 38-kDa human TFIID expressed in bacteria. Taken together, these data suggest that a component of the general transcription machinery, and possibly a TFIID-associated protein, is induced in T cells by PMA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Alternative pathway for induction of human immunodeficiency virus gene expression: involvement of the general transcription machinery. 189 93

Toward gene therapy for the treatment of human immunodeficiency virus type 1 (HIV-1) infections in AIDS, Moloney murine leukemia virus-derived retroviral vectors were engineered to allow constitutive and tat-inducible expression of an HIV-1 5' leader sequence-specific ribozyme (Rz1). These vectors were used to infect the human CD4+ lymphocyte-derived MT4 cell line. The stable MT4 transformants expressing an HIV-1 RNA-specific ribozyme, under the control of the herpes simplex virus thymidine kinase (tk) promoter, were found to be somewhat resistant to HIV-1 infection as virus production was delayed. In cells allowing ribozyme expression under control of the simian virus 40 or cytomegalovirus promoter, the rate of HIV-1 multiplication was slightly decreased, and virus production was delayed by about 14 days. The highest level of resistance to HIV-1 infection was observed in MT4 cells transformed with a vector containing a fusion tk-TAR (trans activation-responsive) promoter to allow ribozyme expression in a constitutive and tat-inducible manner; no HIV-1 production was observed 22 days after infection of these cells. These results indicate that retroviral vectors expressing HIV-1 RNA-specific ribozymes can be used to confer resistance to HIV-1 infection.
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PMID:Resistance to human immunodeficiency virus type 1 (HIV-1) infection in human CD4+ lymphocyte-derived cell lines conferred by using retroviral vectors expressing an HIV-1 RNA-specific ribozyme. 189 2

Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction.
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PMID:Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. 189 41

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:RNA binding by the tat and rev proteins of HIV-1. 190 8

Multiple binding of Tat and nuclear protein(s) to HIV-1 TAR RNA appears to be essential for the Tat-mediated trans-activation. As synthetic Tat-(1-47), which lacks the basic domain and does not bind TAR RNA in vitro, efficiently transactivated HIV-1 LTR in HeLa nuclear extracts, we hypothesized that Tat might trans-activate by interaction with TAR RNA via a host nuclear protein. The role of nuclear proteins in Tat-TAR interaction was examined through evaluation of several synthetic Tat peptides for ability to bind TAR RNA in vitro both in the presence and in the absence of HeLa nuclear proteins. Our data show that both Tat-(1-47) and Tat-(1-86) interact with TAR RNA-bound nuclear proteins, leading to dissociation of the nuclear protein-TAR RNA complexes; the N-terminal sequence of Tat appears to be involved in this interaction. Thus, after binding to TAR RNA, Tat can interact with a proximal TAR-bound nuclear protein and the resulting Tat-nuclear protein complex, now displaced from TAR, may initiate a facile and rapid assembly of the RNA polymerase II transcription complex. This study thus recognizes a novel interaction between Tat and a nuclear protein(s). Here we propose that the interaction of Tat with a nuclear protein(s) occurring on TAR RNA may be one of several steps in the mechanism of Tat-mediated trans-activation of the HIV-1 LTR.
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PMID:Synthetic HIV-1 Tat can dissociate HeLa nuclear protein-TAR RNA complexes in vitro: a novel Tat-nuclear protein interaction. 192 18

The TAR element extending from -17 to +80 in the human immunodeficiency virus long terminal repeat (HIV LTR) is required for activation of gene expression by the tat trans-activator protein. TAR RNA forms a stable stem-loop structure, and mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and the bulge element are the major determinants for tat activation. RNA gel retardation analysis demonstrates that both tat and cellular proteins bind to TAR RNA, but the mechanism by which these proteins increase HIV gene expression is unknown. We have fractionated HeLa cell nuclear extracts in an attempt to identify cellular proteins that bind to TAR RNA and are involved in regulating HIV gene expression. RNA gel retardation and UV cross-linking reveal that a cellular protein of 185 kD, which we designate TAR RNA-binding protein 185 (TRP-185), binds with both high affinity and marked specificity to TAR RNA. RNA gel retardation and competition analyses indicate that TRP-185 binding is strongly dependent on the TAR RNA loop sequences. The binding of TRP-185 is modulated by both a set of cellular cofactors and the tat protein. Highly purified preparations of TRP-185 are capable of activating in vitro transcription of wild-type, but not mutated, HIV LTR chloramphenicol acetyltransferase (CAT) constructs. These results characterize a positively acting cellular RNA-binding factor, TRP-185, which is involved in the regulation of HIV gene expression.
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PMID:tat regulates binding of the human immunodeficiency virus trans-activating region RNA loop-binding protein TRP-185. 193 97

Trans-activation of HIV-1 by the Tat protein is mediated through a cis-acting element (TAR) in the viral RNA. In order to obtain further insight into the molecular interactions for trans-activation, a detailed mutational analysis of TAR RNA was carried out. TAR RNA forms a hairpin structure with important sequence elements in the single-stranded bulge- and loop-domains. We found that the sequence of the base-pairs flanking the bulge is critical for Tat-mediated trans-activation. In addition, Tat-response is reduced when the bulge is forced into a base-paired configuration through the introduction of complementary nucleotides on the opposite side of the stem. Thus, the 3-nucleotide bulge and adjacent base-pairs comprise a recognition domain with both sequence- and structure-elements. Accessibility of the loop sequences is also important for Tat function, since base-pairing through the formation of a pseudoknot-like structure does inhibit Tat action. A third critical parameter that influences the magnitude of Tat response is the number of loop nucleotides. Finally, the relative spacing between the loop and the bulge is also important. We introduced additional base-pairs in the stem connecting the two domains. Such mutations progressively decreased the efficiency of Tat induction. Interestingly, activity of the HIV-2 Tat protein did markedly increase on targets with one or two additional basepairs. These results suggest that Tat interacts with a cellular loop-binding protein(s) to increase HIV gene expression.
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PMID:Detailed mutational analysis of TAR RNA: critical spacing between the bulge and loop recognition domains. 195 76

Human immunodeficiency virus type 1 (HIV-1) gene expression is activated by Tat, a virally encoded protein. Tat trans-activation requires viral (trans-activation--responsive; TAR) RNA sequences located in the R region of the long terminal repeat (LTR). Existing evidence suggests that Tat probably cooperates with cellular factors that bind to TAR RNA in the overall trans-activation process. A HeLa complementary DNA was isolated and characterized that encodes a TAR RNA-binding protein (TRBP). TRBP activated the HIV-1 LTR and was synergistic with Tat function.
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PMID:Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. 201 39

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