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 CTL response to HIV-1 is more vigorous than for any known human pathogen and may be a significant factor in preventing the progression to symptomatic disease. T cell lines, generated by non-specific stimulation with PHA and IL-2, may be reproducibly used to identify HIV-1 isolate-invariant epitopes recognized by the CTL of infected individuals. The CTL response in each of 12 infected individuals to envelope and reverse transcriptase (RT) is dominated by the recognition of one or two viral isolate-invariant epitopes. Seven subjects respond to a single gp160 epitope; three subjects recognize 2 gp160 epitopes. There is a significant increase in recognition of epitopes in the C terminal 104 amino acids of gp41 (p less than 0.002); in fact 40% of the subjects that respond to gp160 recognize the C terminal 20-mer. The CTL-mediated lysis of gp160-expressing targets is MHC restricted, but not all individuals that share the same serologically defined class I-restricting element respond to the same epitope. Recognition of the terminal 20mer is restricted by both A30 and B8. The response to RT in six subjects is distributed over the RT protein. The six subjects recognize four separate regions defined by truncated RT-vaccinia recombinants, but none of the subjects' CTL demonstrate significant recognition of the RT epitope identified in H-2k mice and some humans.
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PMID:Cytotoxic T lymphocytes from HIV-1 seropositive individuals recognize immunodominant epitopes in Gp160 and reverse transcriptase. 137 97

Human fetal thymuses were obtained from abortuses of HIV-1 seronegative females. Thymocytes were isolated and cultured for 2 days with PHA. Thereafter, the culture was divided and half of the cells were exposed to the HIV-1 RF isolate for 4 h. After this incubation period, the HIV-1 exposed and nonexposed control cells were cultured in RPMI 1640 supplemented with IL-2 for 30 days and subsequently maintained in RPMI without the addition of growth factors. Long term culture of both HIV-1 exposed and control thymocytes has yielded two cell lines that have been maintained for more than 3 yr without the addition of growth factors. Flow cytometry using mAb that recognize T cell differentiation markers was used to analyze cell phenotypes. The HIV-1 exposed thymocyte cell line (E88/RF) was shown to be HIV-1 infected by p24 ELISA, reverse transcriptase activity, immunocytochemistry, in situ hybridization, polymerase chain reaction, electron microscopy, and to produce infectious particles by a syncytial forming assay. The non-HIV-1-exposed thymocyte cell line (T412) has remained negative by all criteria for HIV-1 infection. Flow cytometry showed the T412 cells to be positive for the T cell markers CD45, CD38, and CD4 but negative for all other markers tested. The E88/RF cells are positive for CD45 and CD38 but negative for CD4 and other markers. These data report the isolation of two human fetal thymocyte cell lines; one uninfected and susceptible to HIV-1 infection, and the other persistently and productively infected with HIV-1 with little cytopathology. These findings suggest that HIV-1 can persistently infect early T cells and may alter T cell differentiation.
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PMID:Persistent productive HIV-1 infection of a CD4- human fetal thymocyte line. 137 48

Eleven (nine CD4+ and two CD8+) protein purified derivative-specific and eight tetanus toxoid-specific T cell clones (TCC), established from the peripheral blood of healthy persons, were cocultured in vitro with irradiated mononuclear cells from patients infected by HIV in the presence of PHA and polybrene. Two weeks post-HIV exposure, all 17 CD4+, but neither of the two CD8+, TCC exhibited integration of HIV in their genoma, as detected by polymerase chain reaction analysis, and released HIV into their supernatants, as detected by measuring both reverse transcriptase activity and p24 Ag. When co-cultured with either autologous or allogeneic B cells, all CD4+ HIV-infected TCC induced the synthesis of extraordinarily high amounts of IgM, IgG, and IgA. In contrast, their noninfected counterparts could provide helper function for Ig synthesis by autologous B cells only in the presence of the specific Ag (or anti-CD3 antibody), and induced allogeneic B cells to synthesize Ig only upon stimulation with anti-CD3 antibody. The supernatants of HIV-infected TCC failed to stimulate Ig synthesis in B cells. More importantly, when HIV-infected clonal T blasts and B cells were cultured in different chambers separated by a millipore membrane, permeable to molecules but not to cells, Ig synthesis did not occur. The Ig synthesis induced by HIV-infected TCC was also markedly inhibited by the addition in culture of either anti-CD4 or anti-LFA-1 antibody. In contrast, HIV-infected TCC maintained their ability to provide helper function for Ig synthesis in the absence of any stimulus, even after fixation with p-formaldehyde. These data demonstrate that in vitro infection with HIV enables human T cells to stimulate Ig synthesis by B cells by an Ag-nonspecific, MHC-unrestricted, contact-dependent mechanism. This may explain, at least in part, the hypergammaglobulinemia and other phenomena related to polyclonal B cell activation frequently seen in HIV-infected persons.
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PMID:In vitro infection with HIV enables human CD4+ T cell clones to induce noncognate contact-dependent polyclonal B cell activation. 167 84

In this study, we have investigated the basic requirements for HIV-1 infection of CD8+ lymphocytes in vitro. Unfractionated PBL obtained from healthy HIV-1 seronegative donors were activated with PHA and infected in vitro with HIV-1LAV. Based on immunofluorescent labeling, the vast majority of cells (85 to 97%) surviving peak virus replication belonged to the CD8+ subset and only a small percentage (0.5 to 1.5%) were CD4+. Amplification of HIV-1 proviral sequences by polymerase chain reaction performed on the sorted surviving CD8+ cells demonstrated that CD8+ cells harbored HIV-1 proviral DNA. In addition, stimulation of these HIV-1-infected, CD8(+)-sorted cells either with PHA or anti-CD2 mAb resulted in induction of virus replication, as measured by reverse transcriptase activity. Electron microscopic analysis of CD8+ cells chronically infected with HIV-1 and stimulated with PHA showed typical virions budding from, and associated with, the surface of cells immunolabeled with gold beads directed toward the CD8 molecule. Infection of CD8+ cells with HIV-1 occurred only when CD4+ cells were present in the PHA-activated lymphocyte population exposed to HIV-1 at the beginning of the culture or when sorted CD8+CD4- lymphocytes were cocultured with autologous sorted CD8-CD4+ cells that had been previously infected with HIV-1. Coculture of these cells with PHA-blasts and incubation of their supernatants with a CD4+ cell line showed that these chronically infected CD8+ cells could spread HIV-1 infection to uninfected CD4+ cells after stimulation with PHA or anti-CD2 mAb. Therefore, these results suggest that the minimal requirement for in vitro infection of CD3+CD8+CD4- lymphocytes is the presence of infected CD4+ cells and that infected CD8+ T lymphocytes can further spread the infection to CD4+ cells.
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PMID:Infection of CD8+ T lymphocytes with HIV. Requirement for interaction with infected CD4+ cells and induction of infectious virus from chronically infected CD8+ cells. 170 90

Human immunodeficiency virus (HIV) IIIB expression and Epstein-Barr virus (EBV) B95.8-induced transformation were studied during coinfection. Coinfection of peripheral blood lymphocyte (PBL) cultures with HIV and EBV resulted in down-regulation of HIV expression. EBV-induced and spontaneous transformation were markedly reduced in PBL cultures exposed to HIV before EBV. On the other hand, transformation was enhanced when PBL cultures were infected with HIV either simultaneous to or after EBV. Reconstitution of EBV-infected B cell cultures with autochthonous T cells demonstrated that HIV-infected T cells had a reduced ability to inhibit EBV-induced transformation. PHA stimulation of HIV-infected T cells eliminated their ability to inhibit EBV-induced transformation. Lymphoblastoid cell lines (LCLs) established from coinfected PBLs expressed B cell markers and were EBV positive, while a large proportion of the LCLs expressed HIV antigens, released reverse transcriptase activity into the supernatant, and produced syncytia when cocultivated with indicator cell line SupT1. HIV provirus could be detected in LCLs established from coinfected cultures by PCR amplification using specific sets of amplimers for gag and env genes of HIV. To more closely examine the role of various cell types in lymphocyte transformation and HIV replication during coinfection, experiments were carried out using subpopulations enriched for either B or T cells. Simultaneous coinfection of purified B cells with EBV and HIV resulted in a marked reduction of HIV expression, whereas EBV-induced transformation was enhanced. In contrast, spontaneous B cell transformation was inhibited by HIV. A proportion of LCLs established from purified B cells coinfected with EBV and HIV expressed HIV antigens, released reverse transcriptase activity, and produced syncytia on SupT1 cells. These results demonstrate that the IIIB strain of HIV and B95.8 strain of EBV can interact during coinfection of B cells to alter the course of virus expression.
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PMID:Altered HIV expression and EBV-induced transformation in coinfected PBLs and PBL subpopulations. 170 29

The genomes of HIV and SIV are complex and contain several accessory genes which modulate viral replication and pathogenicity. One of these genes, vpx, is unique to the HIV-2/SIV group of viruses and encodes a virion-associated protein of unknown function. To examine the function of vpx, we constructed a vpx-deficient HIV-2 proviral clone and characterized its in vitro biological properties. Following transfection into immortalized T-cell lines, vpx-mutant HIV-2 was fully replication competent and exhibited growth kinetics and cytopathic properties equivalent to wild-type HIV-2. In addition, vpx-deficient virions were indistinguishable from wild-type HIV-2 in ultrastructure, composition of major structural proteins, and reverse transcriptase activity. In PHA-stimulated normal peripheral blood mononuclear cells (PBMCs), however, vpx-deficient virus replicated at substantially lower titers and required a 100- to 1000-fold higher inoculum to establish a productive infection. This defect was localized to early events in the viral life cycle since vpx-deficient virus exhibited a 5- to 10-fold reduction in initial (single cycle) viral DNA synthesis following acute infection of primary PBMCs. Paradoxically, in long-term (9-23 months) cultures of immortalized T-cells (SupT1) continuous high level replication of vpx-deficient, but not wild-type, virus was observed, indicating less efficient viral spread and cell killing and a more attenuated phenotype of vpx-deficient HIV-2. Taken together, these results demonstrate that vpx is required for the production of fully infectious and cytopathic HIV-2 virions and that it functions early in the viral life cycle by facilitating viral entry and/or reverse transcription. The pronounced replicative defect of vpx-deficient HIV-2 in primary PBMCs but not in short-term cultures of immortalized T-cell lines emphasizes the need to characterize the properties of nonessential HIV accessory gene products in natural target cells.
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PMID:Human immunodeficiency virus type 2 vpx protein augments viral infectivity. 171 62

Viral isolates were recovered by cocultivation on macrophage colony-stimulatingfactor (MCSF)-treated monocyte target cells from peripheral blood mononuclear cells (PBMCs) in 25 out of 27 patients seropositive or at risk for HIV infection. Frequency of virus recovery was independent of the patient's age, sex, numbers of CD4+ T cells, clinical stage or zidovudine (azidothymidine) therapy. Sixteen out of 19 HIV isolates were serially passaged in MCSF- treated monocytes. Five out of five virus isolates were also passaged in phytohemagglutinin/interleukin-2 (PHA/IL-2)-treated lymphoblasts. In lymphoblasts, no qualitative or quantitative differences were observed between these isolates and human T-cell leukemia virus IIIB (HTLV-IIIB) for (1) release of p24 antigen reverse transcriptase, and infectious virus, (2) induction of typical cytopathic effects (cell syncytia in 3-10% of cells) and cell lysis, (3) frequency of infected cells (5-20% of PBMC) as detected by in situ hybridization for HIV RNA, (4) down-modulation of T cell plasma membrane CD4, and (5) site of progeny virion assembly and budding (plasma membrane only with no intracytoplasmic accumulation of virus). Progeny virus recovered from infected lymphoblasts was fully infectious for other lymphoblasts, but failed to infect MCSF-treated monocytes. Detailed analysis of target cell tropism among HIV isolates showed that HIV isolated in monocytes infected both monocytes and lymphoblasts; progeny virus isolated in lymphoblasts infected only T cells. HIV interacts differently with monocytes and T cells. Understanding this interaction may more clearly define both the pathogenesis of HIV disease and strategies for therapeutic intervention.
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PMID:Macrophage-HIV interaction: viral isolation and target cell tropism. 211 97

A multicenter study was undertaken to determine the sensitivity and reproducibility of markers for human immunodeficiency virus type 1 (HIV-1) viral growth and the effect of various preparations of lymphocytes on the sensitivity of standard and routinely used procedures for HIV-1 isolation. In phase 1, cocultivated culture supernatants obtained from 10 HIV-1 cultures were transported to three Multicenter AIDS Cohort Study (MACS) Virology Laboratories. Three commercial HIV-p24 antigen capture (AC) tests and two reverse transcriptase (RT) assays were used to ascertain the replication of HIV-1. The Du Pont and Abbott AC assays were found to be most sensitive (85-100%), and the RT assay with 24-h incubation period had comparable sensitivity (75-100%). In phase II, the sensitivity of standard cocultivation procedure for HIV-1 isolation was compared using freshly phytohemagglutinin-P (PHA-P)-stimulated, stimulated-frozen, and frozen-thawed and then stimulated normal human peripheral blood mononuclear cells (PBMCs) as cocultivating cells. Blood samples from 13 HIV-1 infected individuals with various CD4+ cell counts were cocultivated in each of the three MACS laboratories using one of the aforementioned normal PBMCs. The PHA-P-stimulated fresh normal PBMC showed a maximum isolation rate of 100% (13 of 13) with an average of 8 days to positivity. This rate of isolation was significantly greater than other rates using any one of the other PBMC preparations. These findings demonstrated that the use of freshly PHA-P stimulated PBMCs maximized HIV-1 isolation from blood when a sensitive HIV-1 p24 AC assay or RT assay with overnight incubation is employed for the detection of HIV in culture supernatant.
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PMID:The effect of fresh lymphocytes on increased sensitivity of HIV-1 isolation: a multicenter study. 211 52

We have investigated the replicative capacity of 14 primary HIV-1 isolates in cultures of normal blood macrophages and PHA-stimulated peripheral blood mononuclear cells (PBMC). All viruses could infect normal macrophages as demonstrated by either reverse transcriptase (RT) activity, p24 antigen assay, or cocultivation with PBMC. One month after infection of macrophages virus could no longer be detected in the culture medium. The cells remained in this nonproductive state for another month. Virus could, however, be recovered by cocultivation with PHA-stimulated PBMC. Such macrophage-passaged virus induced pronounced cell killing with fragmentation and pyknosis and often replicated poorly in PBMC, in contrast to the original isolate. The results indicate that all primary HIV-1 isolates contain virus variants that can infect cells of the monocyte/macrophage lineage. Persistently infected, seemingly nonproducing cells, may serve as infectious reservoirs in the infected individual and spread infection to other susceptible cells over a long period of time. Moreover, the pronounced killing of PBMC by the macrophage-harbored virus may contribute to the deterioration of the immune system.
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PMID:HIV-1 infection of normal human macrophage cultures: implication for silent infection. 237 82

Monocytes were maintained in tissue culture for greater than 3 mo in media supplemented with rCSF-1. These cultures provided susceptible target cells for isolation and propagation of virus from PBMC of HIV-infected patients. HIV isolated into monocytes readily infected other rCSF-1-treated monocytes but only inefficiently infected PHA-stimulated lymphoblasts. Similarly, laboratory HIV strains passaged in T cell lines or virus isolated from patients' leukocytes into PHA-stimulated lymphoblasts inefficiently infected rCSF-1-treated monocytes. Persistent, low-level virion production was detected in macrophage culture fluids by reverse transcriptase activity or HIV antigen capture through 6-7 wk. Marked changes in cell morphology with cell death, syncytia, and giant cell formation were observed in monocyte cultures 2 wk after infection, but at 4-6 wk, all cells appeared morphologically normal. However, the frequency of infected cells in these cultures at 6 wk was 60-90% as quantified by in situ hybridization with HIV RNA probes or by immunofluorescence with AIDS patients' sera. Ultrastructural analysis by EM also showed a high frequency of infected cells; virtually all HIV budded into and accumulated within cytoplasmic vacuoles and virus particles were only infrequently associated with the plasma membrane. Retention of virus within macrophages and the macrophage tropism of HIV variants may explain mechanisms of both virus persistence and dissemination during disease.
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PMID:Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes. 325 26


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