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

We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping. An analysis of mRNAs generated from pHIVTATA-CAT constructs revealed that transcription starts at the transcription start point and no read-through transcripts are generated.
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PMID:pHIVTATA-CAT, a versatile vector to study transcriptional regulatory elements in mammalian cells. 865 39

HIV-1 infection has been documented in rabbits, but infection proceeds slowly in this species. Human and rabbit cell lines were compared in order to identify barriers to efficient HIV-1 infection of rabbit cells. A direct comparison of human and rabbit CD4 as receptor for HIV-1 indicated that the rabbit CD4 homolog did not function well even when expressed by human cells. Examination of viral RNA production indicated that the major HIV transcripts were produced in HIV-infected rabbit cells, but were present at levels significantly lower than those found for human cells. Ability of HIV-1 LTRs to direct protein expression in human and rabbit cells was compared using gene constructs with the chloramphenicol acetyltransferase (cat) gene flanked by HIV-1 LTRs. Chloramphenicol acetyltransferase protein expression was equivalent in rabbit and human cell lines transfected with the HIV-1/CAT constructs and cotransfections with the HIV-1 tat gene led to similar increases in CAT expression. Subsequent transfections with an infectious molecular HIV clone yielded approximately equal levels of HIV protein expression in rabbit and human cell lines, suggesting that major barriers to virus production in rabbit lines exist at steps prior to transcription of the viral genome. Because HTLV-I replicates with high efficiency in rabbit cells, a chimeric virus clone was constructed consisting of the 5' portion of HIV-1 through the nef coding sequence followed by the 3' HTLV-I LTR. Transfection of most rabbit cell lines with the chimera produced levels of p24gag protein higher than those transfected with the parent HIV-1 clone. By contrast, the unmodified HIV clone replicated more efficiently in all human cell lines tested.
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PMID:Replication of HIV type 1 in rabbit cell lines is not limited by deficiencies in tat, rev, or long terminal repeat function. 867 93

In the present study, a CAT assay, a beta-galactosidase assay, and immunofluorescence analysis have been used to study the cellular uptake of the HIV-1 Tat protein. An anti-Tat MAb binding to an epitope comprising both the basic domain and the RGD sequence inhibits trans-activation by exogenous Tat. Two different full-length recombinant Tat proteins were used in these studies. The inhibitory MAb, however, recognized only one of the recombinant Tat proteins. Immunofluorescence analysis demonstrated that only the Tat protein recognized by the inhibitory anti-Tat MAb was taken up by COS and HeLa cells. This indicates that there are conformational differences between the two Tat proteins and that a correct folding of the epitope recognized by the anti-Tat MAb is required for cellular uptake. The recombinant Tat taken up by the cells was distributed between the nucleoli, the nucleoplasm, and along the nuclear membrane. Interactions between Tat and serum components were shown in vitro and also inhibition of trans-cellular trans-activation by fetal calf serum in tissue culture was demonstrated. The specific inhibition of the cellular uptake of Tat by an anti-Tat monoclonal antibody and the blocking of uptake by serum components implies specific binding of Tat to the cell membrane.
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PMID:A monoclonal antibody defines a novel HIV type 1 Tat domain involved in trans-cellular trans-activation. 874 86

Human immunodeficiency virus type 1 (HIV-1) infection is frequently associated with concurrent infection by opportunistic pathogens, against which production of nitric oxide by host macrophages provides a first line of defence. We have investigated whether regulatory HIV-1 proteins, such as Tat, can modulate the activity of the inducible nitric oxide synthase (iNos) gene when expressed in stable transfectant lines of RAW264.7 cells. A bioassay for Tat, based on transactivation of an HIV-1 LTR-CAT reporter gene, allowed selection of Tat-expressing cells. Parental and Tat-expressing macrophages accumulated identical levels of nitrite following lipopolysaccharide (LPS) stimulation. Interferon gamma (IFN-gamma) stimulation however, resulted in reduced levels of nitrite accumulation as a direct consequence of Tat expression. Conditioned media from Tat-expressing cells reduced the level of nitrite accumulation in parental cells following IFN-gamma stimulation but not stimulation with LPS. These results implicate HIV-1 Tat as a modulator of the IFN-gamma-specific signal transduction pathways leading to iNos expression.
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PMID:The human immunodeficiency virus type 1 regulatory protein Tat inhibits interferon-induced iNos activity in a murine macrophage cell line. 876 Apr 10

We report the characterization of a CAAT enhancer-binding protein (C/EBP) (NF-IL6) element encompassing the region from -174 to -166 of the U3 long terminal repeat (LTR) region of HIV-1. This C/EBP cis sequence was found to bind to C/EBPbeta and C/EBPdelta factors in DNA band shift assay. Transfection of NTera-2 cells with a HIV-1-LTR CAT construct (pC15CAT), together with C/EBPbeta or C/EBPdelta expression plasmids showed that C/EBP proteins strongly activated the HIV-1 promoter. Deletions encompassing the C/EBP-binding site resulted in the enhancement of the LTR activation mediated by C/EBP proteins, suggesting that other sequences located 3' to -170 were indeed the target for C/EBP factors. This possibility was confirmed by using the pCD54E9CAT plasmid, in which the NF-kappaB enhancer was inserted 5' to the HIV-1 LTR TATA box. A NF-kappaB1(p50) expression plasmid was also utilized to test for functional co-operation between NF-kappaB and C/EBP factors. We observed that p50 middle dotC/EBPbeta and p50 middle dotC/EBPdelta complexes were generated in tested cells and strongly activated the HIV-1 LTR by binding to the NF-kappaB sequences. The physical association of NF-kappaB1(p50) with C/EBP factors was assayed by direct interaction of in vitro translated p50 proteins with C/EBPbeta or C/EBPdelta produced as glutathione S-transferase fusion proteins. Moreover, p50 middle dotC/EBPbeta complexes were observed in vivo by using DNA affinity studies with biotinylated NF-kappaB oligonucleotides. By using mutant forms of p50 or C/EBPbeta proteins we found that the transactivation of HIV-1 LTR by p50 middle dotC/EBPbeta complexes required the DNA-binding domain of p50 and the transcription activation domain of C/EBPbeta.
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PMID:Regulation of HIV-1 long terminal repeats by interaction of C/EBP(NF-IL6) and NF-kappaB/Rel transcription factors. 879 13

We compared the efficiency of human immunodeficiency virus (HIV-1) vectors that express a marker gene (chloramphenicol acetyltransferase, CAT) using different promoter elements. In one vector, CAT was expressed under the control of an internal murine leukemia virus (MuLV) long terminal repeat (LTR). In other vectors, CAT production was regulated by the HIV-1 LTR; these vectors also contained the HIV-1 tat gene and pol sequences reported to exert cis-acting positive effects on reverse transcription or gene expression. Vectors employing the Tat-driven HIV-1 LTR exhibited up to 500-fold greater CAT expression in Jurkat lymphocytes or human peripheral blood mononuclear cells compared with vectors using the internal MuLV LTR element as a promoter. This difference was not due to improved packaging of the vector RNA into virions, but to an improved level of gene expression in the target cells. Target cell CAT expression was two- to threefold higher for the vector containing the pol sequences and was only slightly less than that seen for a trans-complemented envdeleted provirus. These results indicate that defective HIV-1 vectors with efficiencies of gene transfer and expression comparable with that of HIV-1 itself are feasible.
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PMID:Use of cis- and trans-acting viral regulatory sequences to improve expression of human immunodeficiency virus vectors in human lymphocytes. 880 25

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.
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PMID:Inhibition of HIV-1 expression by HIV-2. 882 54

Previous studies have shown that expression of HIV-1 provirus was enhanced in cells co-infected with the herpes virus and that HSV-1-mediated induction of the HIV-1 provirus expression involved both NF-kappa B-dependent and NF-kappa B-independent mechanisms. Nuclear NF-kappa B complexes could be detected approximately 8 hr after HSV-1 infection. Using NF-kappa B-specific antibodies in a mobility shift assay, we have found that HSV-1 increases binding of p50, p65, and c-rel to the HIV-1 NF-kappa B probe in both Jurkat T cells and NIH/3T3 cells HSV-1 infection also increases transiently the levels of p50 mRNA but an increase in the level of p65 mRNA was not detected. Furthermore, HSV-1 infection induces a rapid degradation of the I kappa B alpha protein. Transfection of HIV-1 LTR CAT into cell lines which overexpressed individual NF-kappa B proteins showed the highest constitutive expression of CAT activity in cells overexpressing p65. Infection with HSV-1 further enhanced the expression of HIV-1 LTR CAT in cell lines producing p52, p100, and c-rel. In contrast, HSV-1 did not significantly enhance the expression of HIV-1 LTR CAT in cell lines overexpressing p105 and 1 kappa B gamma. In the transient expression assay the p65/c-rel heterodimer was the most effective inducer of the HIV-I LTR expression. Thus it appears that p65 plays a limited role in the NF-kappa B-dependent activation of the HIV-1 LTR following HSV-1 infection and that the stimulation is mediated by the p50/p65 and p65/c-rel heterodimers. Thus the magnitude of HIV-1 provirus induction depends on the relative levels of NF-kappa B subunits present in the infected cells.
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PMID:Differential regulation of the HIV-1 LTR by specific NF-kappa B subunits in HSV-1-infected cells. 886 16

The trans-activator protein (Tat) of HIV-1 plays an important role in viral pathogenesis. Since Tat has been shown to alter expression of a number of host cellular genes, we have investigated the role of Tat in modulating gene expression and differentiation in hematopoietic progenitor cells. Tat protein was introduced in K562 cells, a human hematopoietic progenitor cell line, by either scrape-loading onto HeLa (HL)-tat cells or direct electroporation of an affinity-purified glutathione S-transferase (GST)-Tat fusion protein. Under these conditions, butyric acid-induced hemoglobin production in K562 cells was suppressed by 65 and 52%, respectively. However, coculturing with wild-type HeLa cells or electroporation with the control GST protein did not decrease hemoglobin production. To confirm the presence of bioactive Tat protein within K562 cells, the cells were transiently transfected with a pHIV/LTR-CAT prior to the introduction of Tat. A 30- to 40-fold induction in CAT gene expression was observed in the transfected K562 cells, which were either cocultured with HL-tat or were electroporated with GST-Tat. Simultaneous transient transfection of K562 cells with a TAR expression plasmid, to compete for the availability of Tat protein, significantly downregulated the HIV LTR trans-activation by Tat. In addition, overexpression of the TAR RNAs in K562 cells was able to downregulate the suppressive effect of Tat on butyric acid-induced differentiation. RT-PCR analysis of the total RNAs isolated from these cells demonstrated that Tat protein suppressed the butyric acid-induced gamma-globin gene expression by an average of 54% without affecting the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. These data indicate that the viral Tat protein plays a significant role in abrogating erythroid differentiation in K562 cells.
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PMID:Effect of HIV type 1 Tat protein on butyric acid-induced differentiation in a hematopoietic progenitor cell line. 891 78

CD8+ T lymphocytes of HIV-1-infected individuals can efficiently suppress HIV-1 replication in CD4+ T lymphocytes via soluble factors. We compared the effect of CD8+ T cell-derived supernatants on HIV-1 LTR-driven gene expression in T cells and monocytic cell lines. Our results demonstrate that CD8+ T cell supernatants that suppressed HIV-1 LTR-driven gene expression in Jurkat T cells significantly enhanced expression in Tat-activated U38 monocytic cells in the presence and absence of mitogenic stimulation. Examination of a panel of CD8+ T cell-derived supernatants form HIV-infected individuals demonstrated that the extent of enhancement of transcription in U38 cells was mirrored in most cases by a similar level of suppression of transcription in Jurkat T cells. In latently infected U1 cells treated with TNF-alpha, culture with CD8+ T cell supernatants markedly enhanced virus production. In addition, the percentage increase in the enhancement of HIV-1 LTR-driven CAT expression by CD8+ T cell supernatants correlated strongly (r = 0.911) with the level of p24 detected. The level of LTR-mediated gene expression in U38 cells was not influenced by rhMIP-1 alpha rhMIP-1 beta, or rhRANTES over a wide range of chemokine concentration. Treatment of CD8+ T cell supernatant with a combination of antibodies to these chemokines resulted in a further augmentation of LTR-mediated CAT expression in U38 cells. Taken together, these results demonstrate that CD8+ T cell suppressive factors may have opposite effects on HIV-1 LTR-driven gene expression and replication dependent on target cell type and further suggest that the beta-chemokines do not influence HIV-1 LTR-mediated gene expression in monocytic cells.
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PMID:CD8+ T cell supernatants of HIV type 1-infected individuals have opposite effects on long terminal repeat-mediated transcription in T cells and monocytes. 898 29


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