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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Murine monoclonal antibodies (Mabs) to the major core protein p24 of the human immunodeficiency virus type 1 (HIV-1) were tested for their ability to inhibit the replication and spread of the virus in permanent cell cultures (Molt4/8, K37, H9) and in the culture of II-2 stimulated T cells of healthy donors. After addition of ascitic fluid containing monoclonal anti-p24 antibodies or purified anti-p24 antibodies or the respective control to co-cultures of infected and non-infected cells, HIV-1 replication was evaluated by determining the percentage of infected cells and the activity of reverse transcriptase (RT) in cell-free supernatant. In addition, the supernatant's infectivity was determined. FACS analysis demonstrated p24 antigen in about 40% of unfixed HIV-1 infected cells at the cell membrane. Monoclonal anti-p24 antibodies of different epitope specificity added to the cells but not to the virus delayed the spread of HIV-1 infection in permanent cell culture. Furthermore, anti-p24 Mabs inhibited the release of RT-active virus particles by HIV-1 infected cell lines or II-2 stimulated T-lymphocytes, respectively, up to 60%. The mode of action of anti-p24 antibodies after HIV-1 infection is discussed on the basis of the data obtained.
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PMID:Inhibition of HIV-1 infection in vitro by murine monoclonal anti-p24 antibodies. 162 12

Limiting-dilution techniques were employed to derive single-cell clones from U-937 cells that had been chronically infected with human immunodeficiency virus type 1. All clones thus obtained were positive for the presence of viral antigens; however, not all of the clones produced infectious progeny virus, as detected by the presence of reverse transcriptase (RT) activity in culture fluids. Six of these clones were monitored over time to determine whether their phenotype of human immunodeficiency virus type 1 expression was stable. Three clones maintained production of RT activity at a high level and showed a very high percentage of cells positive for viral p24 antigen, as determined by indirect immunofluorescence. The other three clones showed variations in either their levels of RT activity or the number of cells positive for p24, after which they stabilized. Infectious virus could be recovered from only three clones, as assessed by coculture experiments with different cell types. Two other clones were shown to produce noninfectious viruses. Molecular analyses at the DNA, RNA, and protein levels showed extensive variations between the viral isolates recovered from each clone.
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PMID:High frequency of isolation of defective human immunodeficiency virus type 1 and heterogeneity of viral gene expression in clones of infected U-937 cells. 169 Aug 23

The detection of HIV-1 in human peripheral blood lymphocytes is routinely carried out by cocultivation of test cells with normal peripheral blood mononuclear cells (PBMC). The presence of virus is evidenced by cytologic observation of syncytia or by detecting viral reverse transcriptase (RT) and/or p24 antigen in the culture supernatant fluid. Syncytia formation is almost always associated with the presence of virus as measured by RT, although many RT-positive cultures do not form syncytia. As part of a large screening program, we identified three cultures that showed syncytia but were RT negative. The basis for these discrepant observations was contamination of cultures with mycoplasma that interfered with the RT assay and thereby obscured virus detection. Treatment of cultures with BM-cycline removed mycoplasma contamination and restored RT activity. The present findings indicate the need for caution in the interpretation of negative RT results during HIV-1 isolation and especially in cultures that show evidence of syncytia formation.
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PMID:Suppression of HIV-1 reverse transcriptase activity by mycoplasma contamination of cell cultures. 169 26

HIV1 infection of mononuclear phagocytes is now well established, whereas HIV2 infection of these cells is less well documented. In this work, we studied the replication of the HIV2ROD strain in the U-937 promonocytic cell line and compared it with that of the HIV1BRU strain. p24 antigen and RT (reverse transcriptase) activity were assessed at regular intervals in cell-free supernatants, as was the infectiousness of the produced virus. In the case of HIV2, after a phase of high cell mortality, a chronically infected cell population releasing infectious virions was obtained (the infection remained stable after 60 days of culture). By contrast, for a given multiplicity of infection (i.e. 10,000 cpm RT/10(6) cells), HIV1 replication in U-937 cells was only transient (i.e. 14 days), leading to the synthesis of slightly infectious and probably defective viral particles. Abortive infection was finally obtained, as confirmed by a polymerase chain reaction which failed to detect any proviral HIV1 DNA in the cell line. These results indicate a marked difference between HIV1 and HIV2 in their in vitro interaction with mononuclear phagocytes.
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PMID:HIV2 chronic infection of promonocytic cells. 169 23

We have studied the action of ascorbate (vitamin C) on human immunodeficiency virus type 1 (HIV-1), the etiological agent clinically associated with AIDS. We report the suppression of virus production and cell fusion in HIV-infected T-lymphocytic cell lines grown in the presence of nontoxic concentrations of ascorbate. In chronically infected cells expressing HIV at peak levels, ascorbate reduced the levels of extracellular reverse transcriptase (RT) activity (by greater than 99%) and of p24 antigen (by 90%) in the culture supernatant. Under similar conditions, no detectable inhibitory effects on cell viability, host metabolic activity, and protein synthesis were observed. In freshly infected CD4+ cells, ascorbate inhibited the formation of giant-cell syncytia (by approximately 93%). Exposure of cell-free virus to ascorbate at 37 degrees C for 1 day had no effect on its RT activity or syncytium-forming ability. Prolonged exposure of virus (37 degrees C for 4 days) in the presence of ascorbate (100-150 micrograms/ml) resulted in the drop by a factor of 3-14 in RT activity as compared to a reduction by a factor of 25-172 in extracellular RT released from chronically infected cells. These results indicate that ascorbate mediates an anti-HIV effect by diminishing viral protein production in infected cells and RT stability in extracellular virions.
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PMID:Suppression of human immunodeficiency virus replication by ascorbate in chronically and acutely infected cells. 169 93

In the central nervous system of AIDS patients, human immunodeficiency virus (HIV) infects primarily microglia, a cell type of bone marrow origin. Moreover, microglial cells isolated from adult human brain support the replication of macrophage-adapted strains of HIV type 1 (HIV-1) (B.A. Watkins, H.H. Dorn, W.B. Kelly, R.C. Armstrong, B. Potts, F. Michaels, C.V. Kufta, and M. Dubois-Dalcq, Science 249:549-553, 1990). To determine whether the CD4 receptor, which is expressed in brain, mediates the entry of HIV-1 in microglial cells, we analyzed CD4 transcript expression in cultured microglia using highly sensitive polymerase chain reaction detection of cDNAs synthesized from RNA. With this method, CD4 transcripts could be detected in cultured microglia--as well as in various human brain regions and cultured macrophages used as positive controls--along with transcripts for the LDL and Fc receptors which are characteristic of cells of the macrophage lineage. We then attempted to block viral entry into microglial cells using anti-CD4 antibodies or soluble CD4 (sCD4), which recognize binding sites on CD4 and HIV-1 glycoprotein gp120, respectively. Cultures were pretreated with blocking antibodies (Leu-3a, OKT4A) or virus was preincubated with sCD4 prior to infection with HIV-1 strain AD87(M) or BaL. With either viral strain, these treatments resulted in the prevention of infection or significant and dose-dependent reduction in the number of infected cells and in the levels of reverse transcriptase or p24 antigen released in the medium. Thus, brain-derived microglial cells, which are the primary target of HIV-1 infection in the brain, express the CD4 receptor and this receptor is effectively used for viral entry in vitro.
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PMID:Infection of brain microglial cells by human immunodeficiency virus type 1 is CD4 dependent. 170 42

A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS.
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PMID:HIV-1 propagates in human neuroblastoma cells. 170 60

T-cells from human immunodeficiency virus (HIV)-infected patients are characterized by a number of qualitative deficiencies including defective T-cell activation. The latter has previously been shown to be normally regulated by cAMP. In this study the patterns of cAMP and cGMP induction in MT-4 cells following HIV infection were investigated. The MT-4 cells were infected with HIV (strain IIIb) and at selected times postinfection (p.i.), culture supernatants were tested for HIV replication by reverse transcriptase activity or HIV P24 Ag. The cells were also examined for their intracellular levels of cAMP and cGMP by radioimmunoassay. HIV infection was associated with an increase in intracellular levels of cAMP and cGMP. The cAMP was increased 40-fold by Day 8 and cGMP 4-fold by Day 4 Pl. The increase in intracellular levels of the cyclic nucleotides (CN) were virus specific, dependent on virus dosage, genetically conserved among the two fresh patient isolates tested, and were abolished by uv inactivation. An increase in cAMP and cGMP was also observed in other cell lines infected with HIV. The sustained elevation in CN level observed could certainly influence cell activation and HIV replication and may potentially have clinical relevance.
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PMID:Human immunodeficiency virus infection: association with altered intracellular levels of cAMP and cGMP in MT-4 cells. 170 57

Some murine retroviruses exhibit altered release of virus when cells are treated with alpha interferon (IFN-alpha), resulting in the accumulation of intracellular virions in cytoplasmic vacuoles. In studies of the inhibitory effect of IFN-alpha (Wellferon) on acute human immunodeficiency virus type 1 infection of human T-cell lines, we found that in C3 cells, the 50% effective concentration was 9 U/ml and the 90% effective concentration was 310 U/ml. There was no apparent accumulation of intracellular particles detected by p24 antigen levels or by processing the cells for electron microscopy. Extracellular reverse transcriptase activity and p24 levels decreased in parallel with increasing IFN, whereas the intracellular viral proteins decreased only slightly. By electron microscopy, cells treated with higher concentrations of IFN (512 U/ml) disclosed very few particles budding into extracellular spaces; no intracellular particles could be seen, despite nearly normal levels of intracellular viral protein detected by the p24 antigen assay and correct processing detected by Western blot (immunoblot) analysis. Thus in human immunodeficiency virus-infected cells, the major block produced by IFN-alpha appeared to be late in the viral cycle at the morphogenesis stage of virion production. Chronically infected Jurkat cells treated with IFN appeared to be inhibited in growth rate, as virus production decreased proportionally with cell number.
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PMID:Inhibition of human immunodeficiency virus type 1 morphogenesis in T cells by alpha interferon. 170 4

Crude extracts of dried leaves of Hyssop officinalis showed strong anti-HIV activity as measured by inhibition of syncytia formation, HIV reverse transcriptase (RT), and p17 and p24 antigen expression, but were non-toxic to the uninfected Molt-3 cells. Ether extracts from direct extraction (Procedure I), after removal of tannins (Procedure II), or from the residue after dialysis of the crude extract (Procedure III), showed good antiviral activity. Methanol extracts, subsequent to ether, chloroform and chloroform ethanol extractions, derived from procedure I or II, but not III, also showed very strong anti-HIV activity. In addition, the residual material after methanol extractions still showed strong activity. Caffeic acid was identified in the ether extract of procedure I by HPLC and UV spectroscopy. Commercial caffeic acid showed good antiviral activity in the RT assay and high to moderate activity in the syncytia assay and the p17 and p24 antigen expression. Tannic acid and gallic acid, common to other teas, could not be identified in our extracts. When commercial products of these two acids were tested in our assay systems, they showed high to moderate activity against HIV-1. Hyssop officinalis extracts contain caffeic acid, unidentified tannins, and possibly a third class of unidentified higher molecular weight compounds that exhibit strong anti-HIV activity, and may be useful in the treatment of patients with AIDS.
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PMID:Inhibition of HIV replication by Hyssop officinalis extracts. 170 26


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