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)

We constructed a retroviral vector encoding a mutant tRNA(imet) gene followed by a HIV-1 rev-specific antisense sequence in the U3 region of the 3' long terminal repeat (LTR). This Moloney murine leukemia virus (MoMLV)-based double-copy retroviral vector was used to transduce human lymphoblastoid T-cell lines (CEM, Jurkat). In some clonal cell lines the expected short transcript initiated either from the 5' or 3' LTR tRNA-alpha rev gene was not detectable by Northern blot analyses of transduced, G418-resistant cells with an alpha rev-specific oligonucleotide probe. In other clonal cells, neither the short polymerase III transcript nor the full-length genomic polymerase II transcript (containing the 3' LTR tRNA-alpha rev gene) was detectable when compared with the transduced cell pool. Southern blot and DNA-polymerase chain reaction (PCR) analyses specific for the tRNA-alpha rev cassette in the 5' or 3' LTR of the retroviral vector suggested that the transfer of the 3' LTR U3 region to the 5' LTR was incorrect in most proviruses. These data were confirmed by DNA sequence analyses of several clonal lines demonstrating deletions and insertions. In summary, our results indicate that this retroviral vector design with direct repeats flanking the polymerase III transcription unit plus the alpha rev insert is prone to genetic rearrangements and consequently not useful for the development of gene therapy protocols.
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PMID:Genetic instability of a MoMLV-based antisense double-copy retroviral vector designed for HIV-1 gene therapy. 854 53

A hairpin ribozyme targeting the 3' LTR region (9456) of SIVmac238 was cloned into a murine retroviral vector. This target sequence is conserved among various SIV, as well as most HIV-2, strains. The ribozyme cassette is driven from a polymerase III promoter, that of the human tRNAval gene. Hybrid human B-/T-cell lines (CEM/174) were transduced with the retroviral constructs and selected for G418 resistance. Cells stably expressing the 9456 ribozyme exhibited long-term resistance to infection by a pathogenic molecular clone of SIV and two strains of HIV-2. The ribozyme was also able to effectively reduce the proviral DNA burden. Its efficient protection against SIV/HIV-2 infection constitutes an important step toward evaluating ribozyme gene therapy in a primate model.
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PMID:Intracellular immunization against SIVmac utilizing a hairpin ribozyme. 861 96

We had previously shown that chronically infected ACH-2 cells (HIVLAI) could be superinfected with HIVRF, that the frequency of superinfection increased with time, and that the transcription of the superinfecting virus exceeded that of the host HIVLAI provirus. In contrast, ACH-2 cells superinfected with a nef-substituted neomycin-resistant (proNEO) provirus were not detectable by DNA polymerase chain reaction (PCR) until geneticin (G418) was added, suggesting that the ability to propagate progressively in culture may be HIV strain specific. Clonal populations of ACH-2 superinfected with proNEO did not demonstrate preferential transcription of the superinfecting virus. However, clones of ACH-2 superinfected with HIVRF (ACH2/RF) showed a preponderance of HIVRF transcripts similar to that seen in bulk populations. Induction of the superinfecting virus by phorbol ester (PMA) occurred more rapidly than the hose provirus and did not equalize transcriptional activity. PCR-derived long terminal repeat (LTR) fragments and Tat cDNAs from A3.01 cells acutely infected with HIVRF or from ACH-2 cells were sequenced and tested for transactivation. The HIVLAI LTR was two to three times more Tat-responsive than the HIVRF LTR. TatRF was two to three times more transcriptionally active on either LTR than TatLAI. Demethylation with 5-azacytidine did not significantly affect HIV expression from the HIVLAI host provirus of superinfected ACH2/RF cell clones. These data suggest that the mechanism of preferential transcription in HIVRF superinfected ACH2/RF may be attributed to the Tat/TAR axis and the effect of the specific locus of host proviral integration.
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PMID:Transcriptional effects of superinfection in HIV chronically infected T cells: studies in dually infected clones. 867 41

Genetic modification of hematopoietic stem cells with a synthetic "anti-human immunodeficiency virus type 1 (HIV-1) gene" which inhibits replication of HIV-1 may allow production of mature lymphoid and monocytic cells resistant to HIV-1 growth after autologous transplantation. Because productive HIV-1 replication requires binding of the Rev protein to the Rev-responsive element (RRE) within the viral transcripts for the HIV-1 structural proteins, anti-HIV-1 gene products which interfere with Rev-RRE interactions may inhibit HIV-1 replication. One such strategy involves overexpression of the RRE sequences in transcripts derived from retroviral vectors to act as decoys to sequester Rev protein and prevent its binding to the RRE element in HIV-1 transcripts. We developed an in vitro model to test the efficacy of this gene therapy approach in primary human hematopoietic cells. Human CD34+ hematopoietic progenitor cells from normal bone marrow or umbilical cord blood were transduced with retroviral vectors carrying RRE decoy sequences as part of a long terminal repeat-directed transcript expressing the neo gene (L-RRE-neo) or with a control vector expressing only the neo gene (LN). The transduced progenitors were allowed to differentiate into mature myelomonocytic cells which were able to support vigorous growth of the monocytotropic isolate of HIV-1, JR-FL. HIV-1 replication was measured in unselected cell populations and following G418 selection to obtain uniformly transduced cell populations. Inhibition of HIV-1 replication in the unselected cell cultures was between 50.2 and 76.7% and was highly effective (99.4 to 99.9%) in the G418-selected cultures. Progenitors transduced by either the L-RRE-neo vector or the control LN vector were identical with respect to hematopoietic growth and differentiation. These findings demonstrate the ability of an RRE decoy strategy to inhibit HIV-1 replication in primary human myelomonocytic cells after transduction of CD34+ progenitor cells, without adverse effects on hematopoietic cell function.
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PMID:Transduction of human CD34+ hematopoietic progenitor cells by a retroviral vector expressing an RRE decoy inhibits human immunodeficiency virus type 1 replication in myelomonocytic cells produced in long-term culture. 867 58

Transfer of "anti-HIV-1 genes" into hematopoietic stem cells of human immunodeficiency virus-1 (HIV-1)-infected individuals may be a potent therapeutic approach to render mature cells arising from transduced stem cells resistant to the destructive events associated with HIV-1 infection. To determine the feasibility of gene therapy for acquired immunodeficiency syndrome in individuals already infected with HIV-1, granulocyte colony-stimulating factor mobilized peripheral blood CD34+ cells were isolated from HIV-1-infected individuals and transduced with retroviral vectors containing three different anti-HIV-1-genes: the Rev binding domain of the Rev Responsive Element (RRE decoy) (L-RRE-neo), a double hammerhead ribozyme vector targeted to cleave the tat and rev transcripts (L-TR/TAT-neo), and the trans-dominant mutant of rev (M10) (L-M10-SN). As a control, a vector mediating only neomycin resistance (LN) was used. After 3 days of transduction on allogeneic stroma in the presence of stem cell factor, interleukin-6 (IL-6), and IL-3, the cultures were G418-selected, and then challenged with HIV-1(JR-FL) and a primary HIV-1 isolate. Compared with the control cultures, the L-RRE-neo-, L-TR/TAT-neo-, and L-M10-SN-transduced cultures displayed up to 1,000-fold inhibition of HIV-1 replication after challenge with HIV-1(JR-FL) and the primary HIV-1 isolate. Growth of the hematopoietic cells in long-term bone marrow culture was not perturbed by the presence of any of the anti-HIV-1 genes. This study shows that anti-HIV-1 genes can be introduced into CD34+ cells from individuals already infected with HIV-1, and strongly inhibit HIV-1 replication in primary monocytes derived from the CD34+ progenitors.
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PMID:Inhibition of human immunodeficiency virus-1 (HIV-1) replication after transduction of granulocyte colony-stimulating factor-mobilized CD34+ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes. 911 67

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 have used a biological test on the microplates of cellular cultures in order to investigate the toxicity and the antiviral properties against different viruses: defective Moloney Murine Leukemia virus (MoMLV) derived from the SVX shuttle and expressing resistance to the G418 antimitotic, and Human Immunodeficiency Virus (HIV) of a hydroalcoholic extract from Haemanthus albiflos (Amaryllidacae). The toxicity was assessed through coloric test evaluation of fixed cells stained with crystal violet. In a population of NIH 3T3 cells (Fibroblasts mouse), the toxicity found with 2, 7, 14 and 28 microliters/ml of lyophilisat extract corresponding at: 0.23, 0.81, 1.62 and 3.24 mg of plant dry, was 32, 50, 63 and 70% respectively. With regards to the antiviral properties, the plant extracts showed an inhibition of 88% on the formation of G418 resistant 3T3 clones. The assay on HIV infected lymphotic cells (P4) showed an IC50 of 4 microliters/ml for this extract plant. Therefore, the toxic effect was similar to the antiviral response.
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PMID:[Antiviral activity of hydroalcoholic extract from Haemanthus albiflos on the Moloney murine leukemia virus and the human immunodeficiency virus]. 929 69

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.
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PMID:Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection. 938 99

HIV infection alters the cellular uptake of ions and other small molecules. This study was designed to determine whether hygromycin B, a low molecular weight (MW 527) aminoglycoside protein synthesis inhibitor that is normally impermeable to mammalian cells at micromolar concentrations, can selectively inhibit HIV expression and cytopathology. CD4+ T lymphoblastoid cells (H9) and peripheral blood mononuclear cells (PBMCs) were infected with HIV-1, then incubated in medium containing various concentrations of hygromycin B. HIV-1-induced formation of multinucleated giant cells and single cell killing were dramatically reduced in the presence of micromolar concentrations of hygromycin B. Hygromycin B also inhibited HIV-1 production in a dose-dependent manner during acute infection. G418, a larger and more hydrophobic aminoglycoside (MW 692), did not display the same selective inhibition of HIV-1 production as hygromycin B. Relative to mock-infected cells, protein synthesis in acutely infected H9 cells was selectively inhibited by hygromycin B. Hygromycin B also reduced HIV production in PBMCs and in H9 cells persistently infected with HIV. PCR analysis demonstrated that hygromycin B did not inhibit HIV-1 reverse transcription. These results demonstrate that HIV-1 infection renders cells more sensitive to hygromycin B than uninfected cells, and provides support for the hypothesis that HIV-1 induces an alteration of plasma membrane permeability. The HIV-modified cell membrane may be a potential target for antiviral intervention and chemotherapy.
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PMID:Inhibition of HIV type 1 production by hygromycin B. 967 Dec 17

1. The S. cerevisiae alpha-factor prepro leader is functional and is correctly processed in P. pastoris. 2. P. pastoris has a high secretory capacity, but yields can be severely reduced by extracellular proteases. This problem can be reduced by altering the medium composition, e.g., adjusting the pH or by adding casamino acids. 3. A rapid DNA dot-blot technique can be used for mass screening of transformants to obtain high-copy-number, high-expressing strains. 4. For mEGF, which is an efficiently secreted protein, there was a good correlation between gene dosage and yield, and maximum levels were obtained at high copy number. 5. Vectors conferring resistance to G418 have been developed for the selection of high-copy-number transformants. These vectors can also be used to isolate a series of transformants with increasing copy number of optimizing the expression of genes where high copy number may be detrimental. 6. The HIV-1 ENV gene was not expressed in P. pastoris owing to fortuitous termination of transcription within AT-rich regions. This is a species-specific phenomenon, since full-length HIV-1 ENV transcripts are produced in S. cerevisiae. The problem was overcome by synthesizing the relevent portion of the gene with increased GC content. 7. ENV was hyperglycosylated and immunologically inactive when secreted by P. pastoris. The yield was reduced by extracellular proteases, but like mEGF, this could be significantly improved by altering the pH of the culture medium and by adding casamino acids. 8. In single-copy integrants, transcripts from the semisynthetic HIV-1 ENV gene were almost as abundant as endogenous AOX1. Transcript levels increased progressively with increasing copy number, showing that the AOX1 promoter is not greatly limited by the level of trans-activating factors.
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PMID:Expression of EGF and HIV envelope glycoprotein. 968 Jun 42

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