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)

Human immunodeficiency virus type 1 (HIV-1) isolates are genetically so heterogeneous that they must be described in terms of populations of related but distinct genomes called quasispecies. A recent study of the influence of ex vivo culturing on HIV-1 quasispecies demonstrated that usually low-abundance genomes outgrew the more prominent forms. Here it is shown that multiple passages of an HIV-1 isolate on peripheral blood mononuclear cells resulted in the outgrowth of very minor forms. A single passage of equal proportions of supernatants to either of the established lymphocyte and monocyte cell lines Molt-3 and U937-2, respectively, resulted in the isolation of different sets of minor forms. Recombination between component sequences was observed. Extensive and monotonous base substitutions of G----A (G----A hypermutation) were evident in many sequences. A strong preference for the transition within the GpA dinucleotide was observed. Dislocation mutagenesis, in this case, a -1 slippage or dislocation of the primer with respect to the template, during DNA synthesis by the HIV-1 reverse transcriptase would explain this bias. When the consequences of polymerase errors, recombination, hypermutation, and instability are added to the genetic description of HIV-1, the real complexity of this virus starts to become apparent.
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PMID:Selection, recombination, and G----A hypermutation of human immunodeficiency virus type 1 genomes. 200 43

Human immunodeficiency virus type 1 (HIV-1) NEF protein has been demonstrated to be a negative regulator of HIV-1 replication and HIV-1 LTR transcription under transient expression conditions. The difficulty of several laboratories to reproduce these findings led us to reexamine the role of NEF in HIV-1 provirus expression and HIV-1 LTR transcription. Basal transcription from the HIV-1 LTR in the presence of a NEF expression vector was compared to that in the presence of a mutated NEF vector. NEF expression led to a greater than 10-fold repression of LTR transcription under these conditions. HeLa and Jurkat cell lines carrying the nef gene linked to the CMV promoter or the HIV-1 LTR were isolated by coselection for neomycin resistance. Single cell isolates were further selected for the expression of nef transcripts. With the exception of the anti-sense nef cell lines, all the nef cell lines expressed the 27-kDa NEF protein, detectable by immunoprecipitation. NEF+ HeLa cell lines were at least 5-fold less efficient than NEF- HeLa cell lines in transient proviral expression. Provirus expression was also repressed in the NEF+ Jurkat cell lines. TAT-activated LTR transcription from an HIV-1 LTR-linked CAT expression vector was repressed 10-fold in the NEF+ HeLa and NEF+ Jurkat cell lines. When infected with HIV-1, NEF expressing T lymphoid cell lines showed moderate delays in onset and peak of reverse transcriptase production. However, none of these cell lines completely arrested virus replication. Our data confirm a negative regulatory effect of NEF on both virus production and LTR driven CAT expression in the cell lines tested. It is possible that cell specific factors may influence NEF activity.
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PMID:Human immunodeficiency virus type 1 (HIV-1) provirus expression and LTR transcription are repressed in NEF-expressing cell lines. 202 88

Human immunodeficiency virus type 2 (HIV-2)-related viruses were isolated from a Gambian dying of exclusively neurological disease (HIV-2D194) and from an asymptomatic Ghanian (HIV-2D205). Both strains exhibited properties of HIV-1 biological subtype c: they grew slowly and induced few or no syncytia but eventually produced high levels of particle-associated reverse transcriptase in cultures of fresh peripheral blood lymphocytes, and they established stable infection of T-lymphoma (HUT-78) and monocytic (U937) cell lines. Each produced even higher levels of reverse transcriptase when fresh human monocytes/macrophages were used as target cells. The viruses were molecularly cloned after a single passage in culture, in order to minimize in vitro selection of subtypes present in vivo. Restriction-site analysis showed heterogeneity within each isolate. Nucleotide sequence analysis of a portion of the HIV-2D194 genome revealed that it is a member of the prototypic HIV-2 family, displaying 13% divergence versus HIV-2ROD and HIV-2NIHZ, as compared to 9% divergence between HIV-2ROD and HIV-2NIHZ. In contrast, HIV-2D205 is the most highly divergent HIV-2 strain yet described: it is equidistant in relation between the known HIV-2 strains and the simian immunodeficiency virus isolates from rhesus macaque monkeys (23-25% divergence).
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PMID:Molecular cloning of two west African human immunodeficiency virus type 2 isolates that replicate well in macrophages: a Gambian isolate, from a patient with neurologic acquired immunodeficiency syndrome, and a highly divergent Ghanian isolate. 246 4

Human immunodeficiency virus type 1 (HIV-1) infectivity and cytopathic effect require proper maturation of the viral envelope glycoprotein carbohydrate moieties. We have found that fresh human serum enhances the infectivity of HIV-1 in MT-2 cell infection assays when virus is synthesized in the presence of the mannosidase I inhibitor, 1-deoxymannojirimycin, or the mannosidase II inhibitor, swainsonine, but has no enhancing effect on virus synthesized in the presence of the glucosidase I inhibitors, castanospermine and 1-deoxynojirimycin, or the glucosidase II inhibitor, bromoconduritol. Enhanced infections were characterized by cytopathic effect, antigen synthesis and reverse transcriptase release, all which occurred sooner than in control-infected cultures. This enhancement of infection was also observed in C1q-deficient serum but was not observed in serum that was heat-inactivated or depleted of complement components C3 or factor B, thus suggesting a requirement for the alternate pathway of complement.
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PMID:Antibody-independent, complement-mediated enhancement of HIV-1 infection by mannosidase I and II inhibitors. 247 15

Human immunodeficiency virus type 1 (HIV-1), the etiologic agent of acquired immunodeficiency syndrome (AIDS), has been implicated in the generation of AIDS-associated neurologic dysfunction. We are currently examining the replicative processes involved in HIV-1 infection of selected human fetal neural cell populations in vitro. To determine whether infection of the human fetal dorsal root ganglia (DRG) glial cell population culminates in the production and release of infectious HIV-1, cocultivation and reverse transcriptase (RT) assays were performed. Direct assay of HIV-1 infected neural cell supernatants as well as exposure of permissive SupT1 cells to these HIV-1-infected neural cell supernatants detected no RT activity in either the HIV-1-infected DRG glial cell supernatants or the SupT1 cell supernatants. When SupT1 cells were cocultivated with the HIV-1-infected neural cells for 24-hr intervals, RT activity was detected in the SupT1 supernatants from cocultures initiated less than 2 days after infection (most likely resulting from infectious input virus) but not from cocultures initiated on 3, 5, 10, and 30 days after infection. Hybridization analysis demonstrated transient expression of HIV-1 cytoplasmic mRNA with accumulation reaching a maximum level by 2 to 3 days postinfection, declining thereafter with low, but detectable, levels at 16 days postinfection. In addition, polymerase chain reaction amplification in conjunction with DNA blot hybridization detected HIV-1-specific proviral DNA at 3 days postinfection. Cumulatively, these data suggest that HIV-1 infection of human fetal DRG glial cells culminates in a nonproductive infection with expression of at least a fraction of the virus genome but no detectable infectious virus production.
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PMID:Transient expression of human immunodeficiency virus type 1 genome results in a nonproductive infection in human fetal dorsal root ganglia glial cells. 251 46

Human immunodeficiency virus type 1 (HIV-1) contains an open reading frame called nef at the 3' end of its genome. The nef gene product has been reported to down-regulate viral growth by suppressing viral transcription through interaction with the long terminal repeat region. We have compared two isogenic HIV-1 (HIV-1-WI3) strains, one of which lacks nef expression, and found little difference between them in in vitro growth. We tested effects on viral entry, DNA synthesis, and RNA expression by measuring HIV-specific low molecular weight DNA and RNA after infection. The qualitative and quantitative aspects of DNA and RNA synthesis were comparable between the nef+ and nef- strains. The effects on viral growth were also examined by following changes in reverse transcriptase activity during the course of infection. The presence of the nef gene product failed to slow viral growth in several different cell types tested, including the human T-lymphocyte cell lines H9 and CEM-SS, human primary T cells enriched for CD4+ cells, and human monocytic cell lines U-937 and THP-1. On the contrary, the nef+ strain grew more efficiently in some cell types than the nef- strain. The same results were obtained with nef+ and nef- strains of a different virus, HIV-1-432, whose Nef had been reported to have a negative effect on viral growth. Our data suggest that the Nef protein does not act as a negative factor, at least in the experimental systems employed in our studies.
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PMID:Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. 268 83

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.
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PMID:Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors. 748 5

To determine the effect of disinfectants against viruses in vitro, I devised the Micro-Carrier-Test of dry-fixed virus-infected cells. Human immunodeficiency virus type 1-infected Molt-4 cells (1 x 10(5) cells in 5 microliters of 10% fetal bovine serum) were dry-fixed at the bottom of each well of a 96-well flat-bottomed microtiter plate for 120 minutes at room temperature. Disinfectants were added and allowed to remain for designated times and the wells were washed three times with PBS. Uninfected Molt-4 cells (1 x 10(4) cells/well) were inoculated and cultured for 4 weeks. The culture supernatant was harvested to measure reverse transcriptase activity by non-radioisotopic reverse transcriptase assay every week. Residual cytotoxicity of the disinfectant was determined by cytotoxicity assay. To evaluate the new method, the virucidal efficacy of several well-known disinfectants was reevaluated. Dose- and time-dependent effects of the disinfectants were determined. The minimal effective concentrations after 5 minutes of contact were 20% ethanol, 0.01% glutaraldehyde and 0.1% sodium hypochlorite. These results are almost the same as those reported previously, but there are some discrepancies. The differences between the present and previous protocols are discussed. This Micro-Carrier-Test promises to be useful in the screening of disinfectants.
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PMID:Micro-carrier-test: evaluating disinfectants for HIV. 749 18

Human immunodeficiency virus type 1 (HIV-1) is genetically highly variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombination. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5' ends of multiple RNA and DNA template termini while converting HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT in vitro during polymerization to the 5' ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. HIV-1 RT readily extended recessed DNA primers to form full-length blunt-end DNA-DNA and DNA-RNA duplexes. In addition, HIV-1 RT catalyzed high yields of products with one to four extra nucleotides at the 3' ends of the nascent DNAs. These products were formed processively via a nontemplated mechanism that is highly specific for the addition of purine nucleotides (A > G >> T > or = C). Thus, HIV-1 RT is extremely unfaithful at both DNA and RNA template ends, introducing errors (extra nucleotides) in one out of every two or three nascent strands processively polymerized. This error rate is 1000 times higher than for HIV-1 RT-catalyzed errors at internal template positions. Blunt-end additions were also catalyzed by other retroviral RTs at relative rates of HIV-1 approximately Moloney murine leukemia virus > avian myeloblastosis virus. These data suggest a potentially important mechanism for retroviral mutation mediated by nontemplated blunt-end addition of purines prior to forced copy-choice recombination.
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PMID:Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends. 750 49

Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase (RT) containing lysine (Lys) instead of glutamic acid (Glu) at position 138 proved fully resistant to the inhibitory effect of TSAO derivatives, but retained marked sensitivity to all other HIV-1-specific inhibitors investigated. In contrast, 181 Tyr-->Cys mutated RT lost sensitivity to all HIV-1-specific inhibitors. There was a close correlation between the sensitivity/resistance pattern of HIV-1-specific inhibitors against mutated (138 Glu-->Lys) recombinant HIV-1 RT and mutant virus strains selected for resistance against TSAO-m3T in cell culture and proven to contain the 138-Lys mutation as the sole mutation within the amino acid 50-270 region of their RT.
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PMID:Sensitivity of (138 Glu-->Lys) mutated human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) to HIV-1-specific RT inhibitors. 751 68


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