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)

The expression of the gag-pol polyprotein of human immunodeficiency virus type 1 (HIV-1) occurs via ribosomal frameshifting between the gag and pol genes. Because low levels of the gag-pol precursor are naturally produced in HIV-1-infected cells, a limited amount of information is available on the biology of this molecule. To further study this polyprotein, two mutant HIV-1 proviral genomes were created to position the gag and pol genes in the same translational reading frame. The mutations inserted a single thymidine nucleotide at the site of ribosomal frameshifting (nucleotide 1635), which results in the addition of a phenylalanine residue (frameshift 1 [FS1]), or a single adenine nucleotide, which results in the addition of a leucine residue (frameshift 2 [FS2]). Transfection of the mutant proviral genomes into COS-1 cells resulted in the expression of the p160gag-pol polyprotein precursor as well as the proteolytically processed gag and pol gene products. Metabolic labeling of the transfected cells with [3H]myristic acid revealed that the p160gag-pol and p17gag proteins expressed from the mutant genomes were myristylated. While the supernatants from COS-1 cells transfected with wild-type or mutant proviral genomes contained similar amounts of p24 antigen, the levels of reverse transcriptase were, on the average, 10 times greater in the supernatants from cells transfected with the FS1 and FS2 proviral genomes. The cells transfected with the wild-type proviral genome released infectious viral particles, while the mutant proviral genomes released p24 and reverse transcriptase in the absence of detectable particle formation. The mutant proviral genomes were completely noninfectious as determined by coculture of the transfected COS-1 cells with SupT1 cells. These results demonstrate that the gag-pol polyprotein of HIV-1 contains the appropriate signals for proteolytic processing and association with intracytoplasmic membranes in the absence of virion formation.
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PMID:Overexpression of the gag-pol precursor from human immunodeficiency virus type 1 proviral genomes results in efficient proteolytic processing in the absence of virion production. 187 Feb 15

Two novel enzyme-linked immunoassays (ELISA) for the quantitation of human immunodeficiency virus type 1 (HIV-1) coded glycoprotein with an Mr 120 (gp120) are described. These are based on the highly specific interaction between gp120 and the mannose-specific lectins from Narcissus pseudonarcissus (NPL) and Galanthus nivalis (GNL). Two systems were developed: (1) an HIV-protein ELISA using HIV-protein (also containing HIV-gp120) for the solid phase and NPL as a detector and (2) a lectin-ELISA using the NPL bound to the solid phase and GNL as detector. The HIV-protein ELISA was validated for quantitation of gp120 within the range 3 to 600 ng/ml; the lectin-ELISA for concentrations between 0.6 and 20000 ng gp120/ml. Serum components did not interfere with the binding of gp120 to the lectins. The ELISAs were used for the quantitation of gp120 in HIV-infected CEM cells in vitro. It was found that gp120 appeared in the medium earlier after infection than HIV-p24 and reverse transcriptase, suggesting that gp120 is released as free glycoprotein. Moreover, the ELISAs were also applied successfully for the detection of compounds that bind to gp120 and for the identification of antibodies directed against the highly pathogenic mannan portion of gp120. These ELISAs are considered to be suitable also for the detection of gp120 in the serum of HIV-infected individuals.
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PMID:Human immunodeficiency virus: novel enzyme-linked immunoassays for quantitation of envelope glycoprotein 120. 187 21

An ultrastructural study was performed on rabbit epithelial RK-13 cells and CD4+ human T lymphocyte lines infected with various recombinant vaccinia viruses (RVVs) expressing genes of human immunodeficiency virus (HIV): the mature p17 or p24 gag domain alone, the entire or truncated gag gene, the reverse transcriptase domain, or the gag-pol genes with a frameshift mutation. Cells infected with RVVs that produced the gag polyprotein with a predicted Mr of more than 48K showed budding and release of HIV-like particles into the extracellular space. These particles were not observed in cells expressing a truncated gag gene (p17 and p24 regions). Mature HIV-like particles were observed extracellularly when the entire gag gene and the protease region of the pol gene were expressed. In contrast, in cells infected with RVVs that contained the gag-pol gene with a frameshift mutation, neither recognizable budding structures nor extracellular HIV-like particles could be detected. These results suggest that the gag gene, particularly its 3' terminus, is necessary for the assembly of HIV particles. In addition, the protease region of the pol gene seems to be required for morphological maturation of HIV particles, but complete proteolytic cleavage of the gag protein may prevent bud formation.
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PMID:Role of the gag and pol genes of human immunodeficiency virus in the morphogenesis and maturation of retrovirus-like particles expressed by recombinant vaccinia virus: an ultrastructural study. 191 28

Two naturally occurring lignanolides, isolated from the tropical climbing shrub Ipomoea cairica, (-)-arctigenin and (-)-trachelogenin, were found to inhibit strongly replication of human immunodeficiency virus type 1 (HIV-1; strain HTLV-III B) in vitro. At a concentration of 0.5 microM, (-)-arctigenin and (-)-trachelogenin inhibited the expression of HIV-1 proteins p17 and p24 by 80-90% and 60-70%, respectively. The reverse transcriptase activity in the culture fluids was reduced by 80-90% when the cells (HTLV-III B/H9) were cultivated in the presence of 0.5 microM (-)-arctigenin or 1 microM (-)-trachelogenin. At the same concentrations, the formation of syncytia in the HTLV-III B/H9-Jurkat cell system was inhibited by the compounds by more than 80%. A series of other lignan type compounds displayed no anti-HIV activity. Studying the molecular mechanism of action of (-)-arctigenin and (-)-trachelogenin we found that both compounds are efficient inhibitors of the nuclear matrix-associated DNA topoisomerase II activity, particularly of the enzyme from HIV-1-infected cells. Our results suggest that both compounds prevent the increase of topoisomerase II activity, involved in virus replication, after infection of cells with HIV-1.
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PMID:Differential in vitro anti-HIV activity of natural lignans. 196 81

In contrast to T-cell lines, where CD-4 expression may predict susceptibility to HIV infection, in monocyte hybridomas, presence or absence of surface CD-4 does not appear to be the determining factor of susceptibility to HIV infection. One clone, 20, was documented to be CD-4 negative by surface immunofluorescence as well as by immunoprecipitation. Both CD-4+ and CD-4- human monocyte hybridomas, representative of peripheral blood monocytes were readily infected with HIV (strains IIIB and BR-1 and a variety of patient isolates) as assessed by p24 Ag secretion reverse transcriptase activity and in situ hybridization. Infection occurred in the absence of antibody to HIV suggesting a non Fc mediated process as had been previously described. These data suggest that alternative mechanisms, such as non-specific phagocytosis, may exist for entry of HIV into peripheral blood monocytes. Given these findings, treatment for AIDS, such as the use of soluble CD-4, may not be effective long term, as monocyte infection may still occur and serve as a reservoir for subsequent viral infection of T cells.
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PMID:Surface expression of CD-4 does not predict susceptibility to infection with HIV-1 in human monocyte hybridomas. 196 58

In a survey of 15 different virus isolates, no IFN-alpha or IFN-beta activity was detected in culture fluids of HIV-infected T cells or monocytes. Exogenous rIFN-alpha added to T lymphoblast or monocyte cultures induced restriction in replication of the amphotropic HIV that infect both cell types. With IFN-treated HIV-infected T cells, levels of reverse transcriptase (RT) activity in culture fluids were half those in control cultures, but the frequency of infected cells or the levels of p24 Ag released in culture fluids were unchanged. In contrast to the modest effect of IFN on HIV-infected T cells, IFN-induced antiviral activity in monocytes was quite dramatic. Monocytes treated with IFN at the time of virus challenge showed no evidence of HIV infection: no p24 Ag or RT activity, no viral mRNA, and no proviral DNA. In this system, IFN interrupts one or more early event(s) in the virus replication cycle before formation of proviral DNA. Monocyte cultures infected with HIV 7 days before IFN treatment showed a gradual decrease in levels of p24 Ag and RT activity to baseline by 3 wk. HIV-induced cytopathic changes were markedly reduced, and the frequency of productively infected cells was less than or equal to 1% of total cells. Virus particles released 24 h after IFN treatment were 100- to 1000-fold less infectious than equal numbers of control virions. But, monocytes treated with IFN 7 days after HIV infection were not free of the retroviral pathogen: levels of proviral DNA in the IFN-treated and control HIV-infected cells were indistinguishable. The presence of large quantities of proviral DNA in cells with little or no evidence for active transcription documents a situation approaching true microbiological latency.
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PMID:Regulation of HIV replication in infected monocytes by IFN-alpha. Mechanisms for viral restriction. 197 1

We have performed a prospective 33-month follow-up of the evolution of HIV infection in a cohort of 76 HIV-positive intravenous drug users (IVDUs). We report on immunological and serological variables that proved to be highly predictive of development to AIDS. In a stepwise multivariate analysis of the actuarial progression rate we found the number of CD4+ lymphocytes to be the most powerful predictor of progression to AIDS. We found no independent predictive effects associated with any other variable with predictive power: loss of antibody to p24 antigen, anergy, HIV p24 antigenaemia, loss of antibody to p53 (reverse transcriptase), decreased number of CD8+ T cells, loss of antibody to p31, loss of antibody to p17, beta 2-microglobulin level, loss of antibodies to gp41 and p64, or immunoglobulin A level. We have found that our data differ from those obtained in studies in homosexual men in the different prognostic value of those predictive markers. Our findings should help to identify high risk of progression to clinical AIDS among IVDUs, thereby assisting in the selection of patients for prophylaxis and therapy.
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PMID:Immunological and serological markers predictive of progression to AIDS in a cohort of HIV-infected drug users. 197 43

The virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.
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PMID:Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein. 198 79

An ELISA procedure is described for the quantification of intrathecally synthesized immunoglobulin G antibodies to human immunodeficiency virus (HIV) antigens. Recombinant p17, p24, endonuclease (END), reverse transcriptase (RT), a peptide from the transmembrane region of gp41 (ENV80) and a fusion protein containing HIV-1 and HIV-2 epitopes were compared with a commercially available ELISA. Using a reference serum, antibodies in serum and cerebrospinal fluid (CSF) to all of the antigens could be measured quantitatively in a reliable and reproducible fashion. Despite the fact that the titer varied up to 10(5)-fold between CSF and serum, interassay variability ranged from 3.87% for p17 to 8.41% for RT and intra-assay variability varied from 3.9% +/- 1.2% for p17 to 14.3% +/- 3.9% for the commercial ELISA. Antibody specificity indices (ASI) obtained by relating CSF/serum titers with reference to the corresponding IgG concentrations can be used to detect intrathecal synthesis of virus specific antibodies.
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PMID:The use of recombinant antigens in ELISA procedures for the quantification of intrathecally produced HIV-1-specific antibodies. 199 6

Recombinant full-length CD4 expressed in Spodoptera frugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was "correctly" oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments (125I-CD4 and 51Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, we showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance of HIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cells. RBC-CD4, but not RBCs, aggregated with chronically HIV-1-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.
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PMID:Full-length CD4 electroinserted in the erythrocyte membrane as a long-lived inhibitor of infection by human immunodeficiency virus. 203 80


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