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:1.13.12.7 (firefly luciferase)
2,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.
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PMID:Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1. 1246 32

Lentiviral-mediated gene delivery holds significant promise for sustained gene expression within living systems. Vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 1-based lentiviral vectors can be used to introduce transgenes in a broad spectrum of dividing as well as nondividing cells. In the current study, we construct a lentiviral vector carrying two reporter genes separated by an internal ribosomal entry site and utilize that virus in delivering both genes into neuroblastoma cells in cell culture and into cells implanted in living mice. We utilize two reporter genes, a mutant herpes simplex virus type 1 (HSV1) sr39tk as a reporter gene compatible with positron emission tomography (PET) and a bioluminescent optical reporter gene, firefly luciferase (Fluc), to image expression in living mice by an optical charge-coupled device (CCD) camera. By using this lentivirus, neuroblastoma (N2a) cells are stably transfected and a high correlation (R(2) = 0.91) between expressions of the two reporter genes in cell culture is established. Imaging of both reporter genes using microPET and optical CCD camera in living mice is feasible, with the optical approach being more sensitive, and a high correlation (R(2) = 0.86) between gene expressions is again observed in lentiviral-infected N2a tumor xenografts. Indirect imaging of HSV1-sr39tk suicide gene therapy utilizing Fluc is also feasible and can be detected with increased sensitivity by using the optical CCD. These preliminary results validate the use of lentiviral vectors carrying reporter genes for multimodality imaging of gene expression and should have many applications, including imaging of xenografts, metastasis, and cell trafficking as well as noninvasive monitoring of lentiviral-mediated gene delivery and expression.
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PMID:Noninvasive imaging of lentiviral-mediated reporter gene expression in living mice. 1271 11

Z-100 is an arabinomannan extracted from Mycobacterium tuberculosis that has various immunomodulatory activities, such as the induction of interleukin 12, interferon gamma (IFN-gamma) and beta-chemokines. The effects of Z-100 on human immunodeficiency virus type 1 (HIV-1) replication in human monocyte-derived macrophages (MDMs) are investigated in this paper. In MDMs, Z-100 markedly suppressed the replication of not only macrophage-tropic (M-tropic) HIV-1 strain (HIV-1JR-CSF), but also HIV-1 pseudotypes that possessed amphotropic Moloney murine leukemia virus or vesicular stomatitis virus G envelopes. Z-100 was found to inhibit HIV-1 expression, even when added 24 h after infection. In addition, it substantially inhibited the expression of the pNL43lucDeltaenv vector (in which the env gene is defective and the nef gene is replaced with the firefly luciferase gene) when this vector was transfected directly into MDMs. These findings suggest that Z-100 inhibits virus replication, mainly at HIV-1 transcription. However, Z-100 also downregulated expression of the cell surface receptors CD4 and CCR5 in MDMs, suggesting some inhibitory effect on HIV-1 entry. Further experiments revealed that Z-100 induced IFN-beta production in these cells, resulting in induction of the 16-kDa CCAAT/enhancer binding protein (C/EBP) beta transcription factor that represses HIV-1 long terminal repeat transcription. These effects were alleviated by SB 203580, a specific inhibitor of p38 mitogen-activated protein kinases (MAPK), indicating that the p38 MAPK signalling pathway was involved in Z-100-induced repression of HIV-1 replication in MDMs. These findings suggest that Z-100 might be a useful immunomodulator for control of HIV-1 infection.
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PMID:Inhibition of human immunodeficiency virus type 1 replication by Z-100, an immunomodulator extracted from human-type tubercle bacilli, in macrophages. 1530 54

The impact of cotransfection of mixtures of mutant and wild type (WT) virus on the observed phenotype and replication capacity (RC) in a single-cycle human immunodeficiency virus (HIV) phenotypic assay has been investigated by cotransfecting mutant HIV clones expressing the firefly luciferase expression gene with a WT clone expressing Renilla luciferase. Four mutant constructs with different genotypes displayed <1% RC when transfected alone. Cotransfection of as little as 9% of the WT clone resulted in an 18- to 33-fold increase in the RC of the mutant clones. In addition, the 50% inhibitory concentration (IC(50)) of lopinavir against seven mutant clones decreased by up to 97% after incremental cotransfection of 9 to 50% of the WT clone. The enhancement of RC and decrease in IC(50) for mutant variants following cotransfection with the WT variant appear to be due to complementation rather than genetic recombination. These findings suggest that the RC and susceptibility of plasma isolates from patients who are off therapy or not adherent to treatment, in which WT virus may expand to significant levels, should be interpreted with caution.
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PMID:Complementation in cells cotransfected with a mixture of wild-type and mutant human immunodeficiency virus (HIV) influences the replication capacities and phenotypes of mutant variants in a single-cycle HIV resistance assay. 1536 7

Gene transfer into the central nervous system is an emerging therapeutic strategy for a range of neurological diseases, including neurodegeneration. This approach would benefit from imaging technologies that could determine the extent, magnitude, and duration of transgene expression. We have used bioluminescence imaging (BLI) to image lentiviral vector-mediated gene transfer into the mouse brain. We constructed human immunodeficiency virus type 1 lentiviral vectors that encode firefly luciferase and transduce cells in culture. After stereotactic injection of these vectors into the brain, we were able to detect luciferase expression in living mice and rats. We characterized the signal in mouse brain in terms of localization, kinetics, resolution, and reproducibility and demonstrated that it correlates with the level of firefly luciferase expression. Although the signal decreased gradually to about 20% of the initial value in the first month, the signal remained constant thereafter for more than 10 months. We demonstrated that the light signal can be used as a reporter by using a bicistronic vector. This is the first study to document noninvasive monitoring of long-term transgene expression in the adult mouse brain and provides the basis for applying BLI in the study of brain disease and gene therapeutic strategies.
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PMID:Noninvasive monitoring of long-term lentiviral vector-mediated gene expression in rodent brain with bioluminescence imaging. 1682 Mar 24

Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.
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PMID:Development of a human immunodeficiency virus vector-based, single-cycle assay for evaluation of anti-integrase compounds. 1700 23

Fetal intraperitoneal administration of human immunodeficiency virus (HIV)-l-derived lentiviral vectors (10(7) infectious particles/fetus) has consistently shown high levels of transduction and gene expression in the omentum, peritoneum, and diaphragm when assessed by polymerase chain reaction (PCR) and whole tissue fluorescence. In vivo imaging techniques were explored with early-gestation long-tailed macaques that were administered the vesicular stomatitis virus-glycoprotein (VSV-G)-pseudotyped HIV-1-derived lentiviral vector expressing a mutant herpes simplex virus type 1 thymidine kinase (HSV-1-sr39tk) and firefly luciferase under the control of the cytomegalovirus (CMV) promoter. Fetuses were monitored sonographically and twice during gestation 9-[4-[18F]Fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was injected into the fetal circulation under ultrasound guidance in preparation for microPET imaging. All newborns were delivered at term by cesarean section and raised in the nursery for postnatal studies. At 2 months postnatal age, animals were imaged and biodistribution was assessed. Optical imaging for firefly luciferase expression was also performed every 2 months postnatal age. Under all imaging conditions gene expression was observed in the abdominal region, and closely paralleled findings from prior studies based on whole tissue fluorescence. These investigations have shown that HSV-1-sr39tk and firefly luciferase can be used to safely detect transgene expression at multiple time points in fetal and infant monkeys in vivo and without evidence of adverse effects.
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PMID:Fetal gene transfer using lentiviral vectors: in vivo detection of gene expression by microPET and optical imaging in fetal and infant monkeys. 1713 73

Two new T-cell-based reporter cell lines were established to measure human immunodeficiency virus type 1 (HIV-1) infectivity. One cell line naturally expresses CD4 and CXCR4, making it susceptible to X4-tropic viruses, and the other cell line, in which a CCR5 expression vector was introduced, is susceptible to both X4- and R5-tropic viruses. Reporter cells were constructed by transfecting the human T-cell line HPB-Ma, which demonstrates high susceptibility to HIV-1, with genomes expressing two different luciferase reporters, HIV-1 long terminal repeat-driven firefly luciferase and cytomegalovirus promoter-driven renilla luciferase. Upon HIV infection, the cells expressed firefly luciferase at levels that were highly correlated (r2=0.91 to 0.98) with the production of the capsid antigen p24. The cells also constitutively expressed renilla luciferase, which was used to monitor cell numbers and viability. The reliability of the cell lines for two in vitro applications, drug resistance phenotyping and drug screening, was confirmed. As HIV-1 efficiently replicated in these cells, they could be used for multiple-round replication assays as an alternative method to a single-cycle replication protocol. Coefficients of variation for drug susceptibility evaluated with the cell lines ranged from 17 to 41%. The new cell lines were beneficial for evaluating antiretroviral drug resistance. Firefly luciferase gave a wider dynamic range for evaluating virus infectivity, and the introduction of renilla luciferase improved assay reproducibility. The cell lines were also beneficial for screening new antiretroviral agents, as false inhibition caused by the cytotoxicity of test compounds was easily detected by monitoring renilla luciferase activity.
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PMID:Use of new T-cell-based cell lines expressing two luciferase reporters for accurately evaluating susceptibility to anti-human immunodeficiency virus type 1 drugs. 1718 60

Replication of human immunodeficiency virus type 1 (HIV-1) is controlled by a variety of viral and host proteins. The viral protein Tat acts in concert with host cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR) through a specific interaction with a 59-residue stem-loop RNA known as the trans-activation responsive element (TAR). Inhibitors of Tat-TAR recognition are expected to block transcription and suppress HIV-1 replication. In previous studies, we showed that 2'-O-methyl (OMe) oligonucleotide mixmers containing locked nucleic acid (LNA) residues are powerful steric block inhibitors of Tat-dependent trans-activation in a HeLa cell reporter system. Here we compare OMe/LNA mixmer oligonucleotides with oligonucleotides containing tricyclo-DNAs and their mixmers with OMe residues in four different assays: (1) binding to the target TAR RNA, (2) Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract, (3) trans-activation inhibition in HeLa cells containing a stably integrated firefly luciferase reporter gene under HIV-1 LTR control, and (4) an anti-HIV beta-galactosidase reporter assay of viral infection. Although tricyclo-DNA oligonucleotides bound TAR RNA more weakly, they were as good as OMe/LNA oligonucleotides in suppressing in vitro transcription and trans-activation in HeLa cells when delivered by cationic lipid. No inhibition of in vitro transcription and trans-activation in HeLa cells was observed for tricyclo-DNA/OMe mixmers, even though their affinities to TAR RNA were strong and their cell distributions did not differ from oligonucleotides containing all or predominantly tricyclo-DNA residues. Tricyclo-DNA 16-mer showed sequence-specific inhibition of beta-galactosidase expression in an anti-HIV HeLa cell reporter assay.
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PMID:Tricyclo-DNA containing oligonucleotides as steric block inhibitors of human immunodeficiency virus type 1 tat-dependent trans-activation and HIV-1 infectivity. 1746 63

Liver diseases associated with hepatitis C virus (HCV) infection have become the major cause of mortality in patients with human immunodeficiency virus (HIV) infection since the introduction of highly active anti-retroviral therapy. HCV-related liver disease is more severe in HIV-infected patients than in non-HIV-infected patients, but the standard therapies used to treat chronic hepatitis C in HCV/HIV coinfected patients are the same as those for patients infected with HCV alone. HIV protease inhibitors might have potential to down-regulate HCV load of HCV/HIV coinfected patients. In this study, we evaluated the effects of nelfinavir on intracellular HCV replication using the HCV replicon system. We constructed an HCV replicon expressing a neomycin-selectable chimeric firefly luciferase reporter protein. Cytotoxicity and apoptosis induced by nelfinavir were assessed and synergism between nelfinavir and interferon (IFN) was calculated using CalcuSyn analysis. Nelfinavir dose-dependently repressed HCV replication at low concentrations (IC(50), 9.88 micromol/L). Nelfinavir failed to induce cytotoxicity or apoptosis at concentrations that inhibited HCV replication. Clinical concentrations of nelfinavir (5 micromol/L) combined with IFN showed synergistic inhibition of HCV replication in our replicon model. Our results suggest that the direct effects of nelfinavir on the HCV subgenome and its synergism with IFN could improve clinical responses to IFN therapy in HCV/HIV coinfected patients.
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PMID:Inhibition of intracellular hepatitis C virus replication by nelfinavir and synergistic effect with interferon-alpha. 1930 39


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