Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous reports have demonstrated that the U1 cell line, a model for postintegration latency, is defective at the level of Tat function and can be rescued by exogenously provided Tat protein. Sequence analysis of tat cDNAs from the U1 cell line identified two distinct forms of Tat, in agreement with the fact that this cell line contains two integrated human immunodeficiency (HIV) proviruses. One Tat cDNA lacked an ATG initiation codon, while the other contained an H-to-L mutation at amino acid 13 (H13-->L). Both tat cDNAs were defective in terms of transcriptional activation of long terminal repeat-luciferase reporter gene in transient-transfection experiments. Introduction of the H13-->L mutation in a wild-type tat background caused a severe reduction in transcriptional activation. Introduction of the same mutation in an infectious HIV molecular clone caused a severely defective phenotype which could be rescued when the HIV proviral DNA was transfected in a Jurkat cell line stably expressing the Tat protein (Jurkat-Tat) or in Jurkat cells treated with tumor necrosis factor alpha. Infectious virus stocks generated in Jurkat-Tat cells were used to infect Jurkat cells and exhibited severely impaired growth which could also be rescued by infecting Jurkat-Tat cells. These observations define tat mutations as a mechanism for HIV postintegration latency.
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PMID:Mutations in the tat gene are responsible for human immunodeficiency virus type 1 postintegration latency in the U1 cell line. 944 75

In the human lymphoblastoid T cell line JJhan-5.1, stably transfected with a human immunodeficiency virus-1 long terminal repeat luciferase vector, the level of luciferase activity is dependent on activation of the nuclear factor kappaB (NF-kappaB) transcription factor. Tumor necrosis factor-induced luciferase activity was not modified in JJhan-5.1 cells co-cultivated with murine adenocarcinoma EMT-6 cells but was strongly decreased when nitric oxide (NO) synthase 2 expression was induced in these cells. Two NO synthase inhibitors counteracted this inhibitory effect. Tumor necrosis factor-alpha binding to JJhan-5.1 cells was not modified after incubation with EMT-6 cells. Viability and protein synthesis in JJhan-5.1 cells were also unchanged. Induction of NF-kappaB DNA binding activity was inhibited when EMT-6 cells expressed NO synthase 2 activity. Aminoguanidine, which completely abolished nitrite production, prevented this inhibition. NF-kappaB activation was also strongly inhibited by S-nitrosoglutathione but was marginally affected by N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1, 2-ethylenediamine. Taken together, these results indicated that NO-related species, released by EMT-6 effector cells and probably different from NO itself, inhibited NF-kappaB activation in human lymphoblastoid target cells. Consequently, transcriptional activity of a long terminal repeat-driven luciferase gene construct was markedly diminished.
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PMID:Inhibition of NF-kappaB and HIV-1 long terminal repeat transcriptional activation by inducible nitric oxide synthase 2 activity. 946 73

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated by a host transcription factor, nuclear factor kappaB (NF-kappaB). NF-kappaB belongs to a group of inducible transcription factors and its activity is regulated by multiple cellular signal transduction pathways, including kinases. These kinases are known to be involved in signal-induced NF-kappaB activation and in the induction of HIV-1 gene expression from latently infected cells. In this study we have examined the effect of a newly developed serine/threonine kinase inhibitor, fasudil hydrochloride (FH), on the replication of HIV-1. Although FH was initially developed as a compound that inhibited a myosin light chain kinase (MLCK) and had been approved for clinical use in the treatment of vasospasm after subarachnoid hemorrhage, this study shows its efficacy in blocking HIV-1 replication in latently infected patients. When FH was added to monocytic cell lines latently infected with HIV-1, U1 and OM10.1, the induction of HIV-1 replication by TNF-alpha was blocked at noncytotoxic doses. The IC50 values of HIV-1 induction by FH were 9.3 and 24 microM for U1 and OM10.1, respectively. Because FH could block TNF-alpha-induced, NF-kappaB-dependent gene expression, as examined by the transient luciferase expression assay, the effect of FH was considered to be due to the blocking of the signal transduction pathway of NF-kappaB activation. Although the in vivo effect of FH in blocking HIV-1 induction is not yet known, these findings indicate the feasibility of clinical use of FH and its derivatives in decreasing viral load to prevent clinical development of acquired immunodeficiency syndrome (AIDS) among HIV-1-infected individuals.
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PMID:Inhibition of human immunodeficiency virus type 1 replication by a bioavailable serine/threonine kinase inhibitor, fasudil hydrochloride. 951 89

Rolipram, a phosphosdiesterase type IV-specific inhibitor, prevented p24 antigen release from anti-CD3-activated human immunodeficiency virus (HIV)-infected T cells and CD4(+)-cell depletion associated with viral replication in a dose-responsive manner but minimally inhibited T-cell proliferation. Moreover, rolipram reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10) by HIV-infected T cells. The transcriptional ability of a luciferase reporter gene under control of the HIV long terminal repeat, induced by phorbol myristic acetate plus ionomycin or by TNF-alpha, in primary T and Jurkat cells was also inhibited by rolipram. Rolipram inhibited NF-kappaB and NFAT activation induced by T-cell activation in Jurkat and primary T cells, as measured by transient transfection of reporter genes and electrophoretic mobility shift assays. Exogenous addition of TNF-alpha in the presence of rolipram restored NF-kappaB but not NFAT activation or p24 release. Addition of dibutyryl-cyclic AMP (dBcAMP) mimicked the effects of rolipram on p24 antigen release, NF-kappaB activation, and TNF-alpha secretion, but it did not affect NFAT activation or IL-10 production. The protein kinase A inhibitor KT5720 prevented the inhibition of TNF-alpha secretion but not that of HIV type 1 (HIV-1) replication caused by rolipram. Our data indicate that blockade of phosphodiesterase type IV could be of benefit against HIV-1 disease by modulating cytokine secretion and transcriptional regulation of HIV replication, and they suggest an important role of NFAT in HIV replication in primary T cells. Some of those activities cannot be ascribed solely to its ability to increase cAMP.
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PMID:Inhibition of phosphodiesterase type IV suppresses human immunodeficiency virus type 1 replication and cytokine production in primary T cells: involvement of NF-kappaB and NFAT. 957 35

Tuberculosis has emerged as an epidemic, extended by the large number of individuals infected with human immunodeficiency virus type 1 (HIV-1). The major goal of this study was to determine whether the mycobacterial cell wall component mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis (M. tuberculosis) could activate transcription of HIV-1 in T cells with the use of an in vitro cell culture system. These experiments are of prime importance considering that CD4-expressing T lymphocytes represent the major virus reservoir in the peripheral blood of infected individuals. Using the 1G5 cell line harbouring the luciferase reporter gene under the control of the HIV-1 LTR, it was first found that culture protein filtrates (CFP) from M. tuberculosis or purified ManLAM could activate HIV-1 LTR-dependent gene expression unlike similarly prepared CFP extracts devoid of ManLAM. The implication of protein tyrosine kinase(s), protein kinase A and/or protein kinase C was highlighted by the abrogation of the ManLAM-mediated activation of HIV-1 LTR-driven gene expression using herbimycin A and H7. It was also determined, using electrophoresis mobility shift assays, that M. tuberculosis ManLAM led to the nuclear translocation of the transcription factor NF-kappaB. M. tuberculosis ManLAM resulted in clear induction of the luciferase gene placed under the control of the wild-type, but not the kappaB-mutated, HIV-1 LTR region. Finally, the ManLAM-mediated activation of HIV-1 LTR transcription was found to be independent of the autocrine or paracrine action of endogenous TNF-alpha. The results suggest that M. tuberculosis can upregulate HIV-1 expression in T cells and could thus have the potential to influence the pathogenesis of HIV-1 infection.
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PMID:Mycobacterium tuberculosis mannose-capped lipoarabinomannan can induce NF-kappaB-dependent activation of human immunodeficiency virus type 1 long terminal repeat in T cells. 963 75

The ICAM-1/LFA-1 interaction has been clearly demonstrated to play an active role in syncytium formation induced by human immunodeficiency virus type 1 (HIV-1). Since it is known that a high-affinity state of LFA-1 for ICAM-1 can be induced through conformational change, such a high-affinity state may also contribute to the process of syncytium formation. In this study, we have investigated the involvement of the conformational status of LFA-1 in HIV-1-dependent syncytium formation by using the anti-LFA-1 antibody NKI-L16, which is known to activate the high-affinity state. Initial visual observations by light microscopy indeed suggested that the addition of the NKI-L16 antibody led to bigger and more numerous syncytia when different cell lines were tested. To further analyze this NKI-L16-dependent increment of syncytium formation in a quantitative assay, a new luciferase-based assay was developed by using a T-cell line containing an HIV-1 long terminal repeat (LTR)-driven luciferase construct (1G5) in coincubation with an HIV-1-positive cell line (J1.1). Upon fusion, the viral Tat protein could diffuse to the 1G5 cells, leading to a transcriptional increase of the HIV-1 LTR-driven luciferase gene. Initial evaluation of this assay showed a good correlation between the level of syncytium formation determined by microscopic observation and the level of measured luciferase activity. In addition, this assay showed a greater induction of enzymatic activity correlating with syncytium formation in comparison to a similar incubation with the HeLa-CD4-LTR-beta-gal indicator cell line. By using this test, NKI-L16 treatment of 1G5/J1.1 cells led to a three- to sevenfold increase in HIV-1 LTR-driven luciferase activity. The syncytium-dependent luciferase activity in NKI-L16-treated cells could be blocked by classical syncytium inhibitors such as soluble CD4, anti-CD4, and anti-gp120 antibodies. Inhibition could also be observed with specific blocking agents for the chemokine receptor CXCR4, as well as with soluble ICAM-1, anti-LFA-1, anti-ICAM-1, and anti-ICAM-2 blocking antibodies, indicating the requirement for the LFA-1/ICAM interaction. Treatment of peripheral blood mononuclear cells with NKI-L16 resulted in a higher level of syncytium formation in the presence of the cell line J1.1. Conversely, when PBMCs were infected with two different syncytium-inducing HIV-1 primary isolates, coincubation with NKI-L16-pretreated 1G5 cells led to higher levels of luciferase activity for both virus isolates. Our results therefore show for the first time a direct role for the LFA-1 high-affinity state in virus-mediated syncytium formation. Based on the demonstration that an increase in ICAM-1 binding is induced by T-cell activation, these data suggest an in vivo involvement of the high-affinity state of LFA-1 in HIV-1-induced syncytium formation. Moreover, syncytia might preferentially occur in lymph nodes, since this microenvironment harbors a high proportion of activated T cells.
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PMID:Modulation of human immunodeficiency virus type 1-induced syncytium formation by the conformational state of LFA-1 determined by a new luciferase-based syncytium quantitative assay. 969 6

The US22 gene family was first discovered in human cytomegalovirus (HCMV) and contains several conserved amino acid motifs. Human herpesvirus 6 (HHV-6) also encodes several genes belonging to the US22 family, including the U3 gene (a positional homolog of HCMV UL24). Because the gene products of the US22 gene family function as gene regulators in general, we analyzed the HHV-6A U3 gene. Six transcripts with different molecular weights of 7.5-1.8 kb were detected by Northern blot analysis using a U3-specific probe. Sequence analyses of the respective cDNA clones and primer extension experiments revealed that the U3 gene encoded the 2.0- and 3.5-kb transcripts and had no splicing within the U3 gene region. By immunofluorescence testing using antibodies raised to a fusion protein of MBP (maltose-binding protein) and U3, the U3 viral antigen was detected as early as 24 h p.i. in HHV-6A-infected U373 cells. The antigens were found in cytoplasmic granules, preferentially in the endoplasmic reticulum. Moreover, cotransfection assay using a luciferase gene expression system revealed that the U3 gene product was capable of activating the human immunodeficiency virus long terminal repeat promoter in CV1 cells.
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PMID:Analysis of human herpesvirus 6 U3 gene, which is a positional homolog of human cytomegalovirus UL 24 gene. 974 Jul 84

Replication of human immunodeficiency virus type-1 (HIV-1) is highly dependent on the state of activation of the infected cells and is modulated by interactions between viral and host cellular factors. Prostaglandin E2 (PGE2), a pleiotropic immunomodulatory molecule, is observed at elevated levels during HIV-1 infection as well as during the course of other pathogenic infections. In 1G5, a Jurkat-derived T cell line stably transfected with a luciferase gene driven by HIV-1 long terminal repeat (LTR), we found that PGE2 markedly enhanced HIV-1 LTR-mediated reporter gene activity. Experiments have been conducted to identify second messengers involved in this PGE2-dependent up-regulating effect on the regulatory element of HIV-1. In this study, we present evidence indicating that signal transduction pathways induced by PGE2 necessitate the participation of cyclic AMP, protein kinase A, and Ca2+. Experiments conducted with different HIV-1 LTR-based vectors suggested that PGE2-mediated activation effect on HIV-1 transcription was transduced via both NF-kappaB-dependent and -independent signaling pathways. The involvement of NF-kappaB in the PGE2-dependent activating effect on HIV-1 transcription was further confirmed using a kappaB-regulated luciferase encoding vector and by electrophoretic mobility shift assays. Results from Northern blot and flow cytometric analyses, as well as the use of a selective antagonist indicated that PGE2 modulation of HIV-1 LTR-driven reporter gene activity in studied T lymphoid cells is transduced via the EP4 receptor subtype. These results suggest that secretion of PGE2 by macrophages in response to infection or inflammatory activators could induce signaling events resulting in activation of proviral DNA present into T cells latently infected with HIV-1.
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PMID:Prostaglandin E2 Up-regulates HIV-1 long terminal repeat-driven gene activity in T cells via NF-kappaB-dependent and -independent signaling pathways. 976 56

Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1beta and SDF-1alpha, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.
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PMID:Microglia express CCR5, CXCR4, and CCR3, but of these, CCR5 is the principal coreceptor for human immunodeficiency virus type 1 dementia isolates. 984 23

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) introduced in association with the luciferase reporter gene into Jurkat T cells was strongly activated by a combination of human neutrophils and phorbol myristate acetate (PMA). Activation was not observed when normal neutrophils were replaced by neutrophils which lack a respiratory burst, ie, from a patient with chronic granulomatous disease (CGD), was strongly inhibited by catalase, was potentiated by vanadate, was stimulated by relatively low concentrations of azide, and was inhibited by selective inhibitors of protein kinase C (PKC). The PMA affected activation in three ways: (1) by directly activating the LTR in Jurkat LTRluc; (2) by inducing a respiratory burst in neutrophils with the formation of H2O2; and (3) by increasing the sensitivity of Jurkat LTRluc to the activating effect of H2O2. When PMA was replaced by opsonized zymosan as the neutrophil stimulus, activation of the LTR was low unless azide was added. Activation in the presence of azide was not seen when CGD neutrophils were used or when catalase was added, suggesting that azide acts by inhibiting the degradation of H2O2. These findings indicate that activation of the HIV-1 LTR in Jurkat T cells can be induced by H2O2 released by neutrophils, particularly when PKC is concomitantly activated.
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PMID:Activation of the human immunodeficiency virus-1 long terminal repeat by respiratory burst oxidants of neutrophils. 986 80


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