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

HIV-1 transcription from the LTR promoter is activated by the viral Tat protein through interaction with the nascent TAR RNA hairpin structure. The mechanism of Tat-mediated transcriptional activation has been extensively investigated with LTR-CAT reporter genes in transient transfections and, more recently, in infection experiments with mutant HIV-1 variants. Several discrepancies between these two assay systems have been reported. For instance, whereas opening of the lower part of the TAR RNA stem does not affect the promoter activity of an LTR-CAT plasmid in transient assays, the corresponding virus mutant is fully replication-impaired. With the aim to resolve this controversy, we have examined the activity of a set of TAR RNA mutants in transient transfection experiments with a variety of cell types. We now demonstrate that truncated TAR motifs exhibit a severe, but cell-type dependent transcription defect. Whereas full LTR activity is measured in COS cells that have been used regularly in previous transfection assays, a severe defect is apparent in a variety of human cell lines, including T cell lines that are typically used in HIV-1 replication studies. These results suggest the presence of a human protein that participates in Tat-mediated transcriptional activation through binding to the lower part of the TAR stem. Several candidate co-factors have been reported in literature. This study resolves the discrepancy between transfection and infection studies on the requirements of the lower TAR stem structure. The evidence also implies that LTR transcription studies should be performed preferentially in human cell types.
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PMID:Optimal Tat-mediated activation of the HIV-1 LTR promoter requires a full-length TAR RNA hairpin. 901 87

The human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein is essential for efficient viral gene expression and virus replication. The Tat core domain, a stretch of 12 amino acids between the cysteine-rich and the basic domain, is conserved in all HIV isolates and required for interaction with a number of cellular transcriptional regulatory proteins. Here we demonstrate that soluble peptide analogs of the Tat core domain (amino acid 36-50) are able to effectively block LTR transactivation. In transfection experiments, Tat core peptide analogs containing amino acid substitutions at position 41 and 44 inhibited Tat transactivation of an HIV-1 LTR-CAT reporter construct up to 80-fold. In contrast, inhibition of other promoters such as HTLV-I and CMV was approximately 2-fold. Tat peptide analog 36-50 (41/44) inhibited HIV virus replication by 85% in latently infected U1 cells induced with Tat. Furthermore, U1 cells treated with the Tat peptide 36-50 (41/44) analog showed markedly delayed virus transmission when cocultivated with parental U937 cells. Interestingly, while both short and long peptide analogs (amino acids 36-50 vs 36-72) inhibited Tat transactivation in transient assays, the short peptides were more effective inhibitors of virus replication in U1 cells. The Tat peptide analog did not decrease expression of cellular genes including beta-actin, GAPDH, and histone H2B.
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PMID:Inhibition of HIV-1 transcription and virus replication using soluble Tat peptide analogs. 901 42

Using HeLa cells stably transfected with an HIV-LTR-CAT construct, we demonstrated a peak in CAT induction that occurs in viable (but not necessarily cell-division-competent) cells 24 h following exposure to some cell-killing agents. gamma rays were the only cell-killing agent which did not induce HIV transcription; this can be attributed to the fact that gamma-ray-induced apoptotic death requires functional p53, which is not present in HeLa cells. For all other agents, HIV-LTR induction was dose-dependent and correlated with the amount of cell killing that occurred in the culture. Doses which caused over 99% cell killing induced HIV-LTR transcription maximally, demonstrating that cells that will go on to die by 14 days are the cells expressing HIV-LTR-CAT.
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PMID:HIV expression is induced in dying cells. 911 23

Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5' deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5'-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stem-loop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBPbeta, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.
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PMID:HIV-1 Tat induces the expression of the interleukin-6 (IL6) gene by binding to the IL6 leader RNA and by interacting with CAAT enhancer-binding protein beta (NF-IL6) transcription factors. 916 58

The LTRs of HIV-1 and HTLV-I have been shown by several laboratories to be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). This agent is a potent activator of protein kinase C (PKC). However, long exposure to TPA downregulates PKC in many cell types. We demonstrated that TPA treatment of Jurkat cells for more than 24 hr resulted in a sever depletion of this enzyme. Therefore, to explore the role of PKC in the effect of TPA on these LTRs, we transfected Jurkat cells with HIV-1 LTR-CAT or HTLV-I LTR-CAT construct after 72 hr of TPA pretreatment. While this TPA pretreatment considerably reduced the HIV-1 LTR basal expression, it strongly stimulated the expression of HTLV-I LTR. Furthermore, when TPA was added after transfection, a strong stimulation of HIV-1 LTR was observed, which could be abrogated by PKC inhibitors like H7 and chelerythryn. However, under these conditions TPA stimulated HTLV-I LTR to a lesser extent than did the long-term TPA pretreatment. Moreover, this stimulation was enhanced by the PKC inhibitors. Thus our data indicate that while the effect of TPA on HIV-1 LTR is strictly dependent on PKC activity, its effect on HTLV-I LTR is exerted via a different pathway that not only does not require PKC activation but rather seems to be antagonized by the activated PKC. Using a deletion mutant of HTLV-I LTR we mapped the PKC-independent effect of TPA to the c-ets responsive region 1 (ERR-1) located in U3 of this LTR.
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PMID:The long terminal repeats of human immunodeficiency virus type-1 and human T-cell leukemia virus type-I are activated by 12-O-tetradecanoylphorbol-13-acetate through different pathways. 919 47

Transcription of the human immunodeficiency virus type 1 (HIV-1) genome takes place after integration of the provirus into human chromosomal DNA. HIV transcription is known to be modulated by viral and cellular factors but the influence of flanking chromosomal sequences on proviral gene expression has not been well defined. To investigate the activity of the integrated HIV promoter, we exploited the ability of recombinant adeno-associated virus (AAV-2) to transfer and stably integrate genes into the human genome at random or site-specifically. Chimeric AAV vectors were constructed containing an HIV-CAT reporter cassette; some vectors also contained the neomycin resistance gene to facilitate the isolation of positive clones. HeLa cells were infected with recombinant AAV, in some instances together with wild-type virus as a source of AAV rep function. We isolated 25 clones of G418-resistant cells which carried the integrated HIV-CAT cassette, generally occupying unique sites that did not correspond to the AAV-specific region of chromosome 19. The HIV promoter was transcriptionally active in most of the clones. Basal promoter activity varied substantially among the clones, and its responsivity to the HIV transactivator Tat was also variable. The integrated HIV promoter was transactivated to comparable degrees by the one-exon form and two-exon form of Tat. These findings provide evidence that the transcriptional activity of the HIV promoter can be greatly influenced by the site of proviral insertion.
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PMID:Transduction of the human immunodeficiency virus type 1 promoter into human chromosomal DNA by adeno-associated virus: effects on promoter activity. 923 45

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 efficiency 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 efficient 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.
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PMID:Sequence elements downstream of the human immunodeficiency virus type 1 long terminal repeat are required for efficient viral gene transcription. 929 45

Previously we documented the transposition of an intracisternal A particle (IAP) provirus to the interleukin 3 (IL-3) locus which resulted in autocrine transformation. In the present study, the effects of different long terminal repeats (LTRs) on IL-3 gene expression and autocrine transformation were investigated. LTRs from defective IAPs, and replication competent Moloney murine leukemia virus (MoMuLV), human T cell leukemia (HTLV), and immunodeficiency (HIV) viruses, were inserted 5' of the IL-3 promoter region, and their transforming abilities determined. Addition of the lymphocyte specific (LS) IAP-LTR to the germline IL-3 (gIL3) gene, the IAP-LTR present in the previously described transposition, resulted in a modified IL-3 gene that only infrequently transformed IL-3-dependent cells. In contrast, addition of plasmacytoma (PC) IAP-LTRs to the gIL3 gene, which were isolated from IAPs expressed in plasmacytomas, resulted in modified IL-3 genes that transformed IL-3-dependent cells more readily. The MoMuLV-LTR and the TCRdelta enhancer also stimulated high levels of IL-3 expression and autocrine transformation. In contrast, the HTLV-I, HTLV-II and HIV LTRs did not induce significant IL-3 synthesis or autocrine transformation. Consistent with these results, higher levels of CAT expression were observed in cells transiently transfected with PC-IAP-LTR or a TCR enhancer compared with LS-IAP and HTLV LTRs. In summary, the rank order for the effects of different LTRs on IL-3 expression and cell transformation is: TCRdelta-enhancer approximately MoMuLV-LTR > PC-IAP-LTRs >> LS-IAP-LTR >> HTLV-LTRs approximately HIV-LTR. These results indicate that the LS-IAP-LTR is very weak at inducing IL-3 gene transcription and additional genetic mutations may be necessary for LS-IAPs to induce autocrine transformation of hematopoietic cells. In contrast, the enhancers contained in PC-IAP-LTRs and TCR enhancers may be more effective in inducing abnormal gene expression and malignant transformation.
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PMID:Differential effects of retroviral long terminal repeats on interleukin-3 gene expression and autocrine transformation. 932 93

The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent trans-activator of transcription from the viral LTR promoter. Previous mutagenesis studies have identified domains within Tat responsible for binding to its TAR RNA target and for transcriptional activation. The minimal Tat activation domain is composed of the N-terminal 48 residues, and mutational analyses identified a cluster of critical cysteines. The importance of four highly conserved aromatic amino acids within the activation domain has not been thoroughly investigated. We have systematically substituted these aromatic residues (Y26, F32, F38, Y47) of the HIV-1 LAI Tat protein with other aromatic residues (conservative mutation) or alanine (nonconservative mutation). The activity of the mutant Tat constructs was measured in different cell lines by transfection with a LTR-CAT reporter plasmid. The range of transcriptional activities measured for this set of Tat mutants allowed careful assessment of the level of Tat activity required for optimal viral replication. To test this, the mutant Tat genes were introduced into the pLAI infectious molecular clone and tested for their effect on virus replication in a T-cell line. We found that a twofold reduction in Tat activity already affects viral replication, and no virus replication was measured for Tat mutants with less than 15% activity. This strict correlation between Tat activity and viral replication demonstrates the importance of the Tat function to viral fitness. Interestingly, a less pronounced replication defect was observed in primary cell types. This finding may correlate with the frequent detection of proviruses with Tat-inactivating mutations in clinical samples.
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PMID:Determination of the minimal amount of Tat activity required for human immunodeficiency virus type 1 replication. 935 35

In HIV-1 infection, Tat acts at least in part to control transcriptional elongation by overcoming premature transcriptional termination. In some other genes this process is governed by DNA elements called attenuators in concert with cellular transcription factors. To understand the action of Tat more fully and explore its role as an anti-attenuator, we examined the ability of several natural and synthetic attenuation sequences to modulate transcription initiated at the HIV LTR. Fragments containing these signals were inserted downstream of the TAR element in an HIV-CAT chimera and their effects on transcription were assessed both in vitro and in vivo. Runoff transcription assays in HeLa cell extracts demonstrated that the attenuators give rise to premature termination of transcripts initiated from the heterologous HIV-LTR promoter in vitro. When transiently expressed following transfection into Cos cells, however, premature transcript termination at the attenuation site was not observed. Nevertheless, many of the inserted sequences exerted marked effects on CAT gene expression and on transactivation by Tat at both the RNA and protein levels. The nature and magnitude of the effects depended upon the identity of the attenuator and its orientation but only one of 16 sequences tested met the criteria for a Tat-suppressible attenuator in vivo. One other sequence, in contrast, severely reduced Tat-activated transcription without inhibiting basal transcription These results indicate that sequences downstream of the HIV LTR can influence its function as a promoter and its response to Tat transactivation, but lend little support to their role as attenuators in vivo.
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PMID:Effects of heterologous downstream sequences on the activity of the HIV-1 promoter and its response to Tat. 939 10


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