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)

Recently, several classes of compounds have been shown to be extremely selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. These include the tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO), 1-(2-hydroxyethoxymethyl)-6-(phenylthio)-thymine (HEPT), dipyridodiazepinone, pyridinone and bis(heteroaryl)piperazine derivatives. The hallmark of these new antiviral compounds is a specific interaction with reverse transcriptase (RT) of HIV-1. They are inactive against HIV-2 and any other viruses tested. Here we describe that, in addition to the HIV-1 strains, two simian immunodeficiency virus (SIV) strains from African green monkeys (SIVagm3 and SIVagmTYO-1) are also sensitive to the TIBO class of compounds. TIBO and HEPT derivatives block the replication of SIVagm in cell culture at micromolar concentrations. Kinetics of inhibition of SIVagm RT by TIBO are competitive with respect to the natural substrate (dGTP). Amino acid alignments and site-directed mutagenesis point to the critical role of amino acid residues Y181 and Y188 in the sensitivity of HIV-1 RT and SIVagm RT to inhibition by the TIBO derivatives. Antiviral efficacy studies with this range of compounds and using sensitive SIV strains are now feasible in monkeys.
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PMID:Differential inhibitory effects of TIBO derivatives on different strains of simian immunodeficiency virus. 137 81

The gastrointestinal tract is considered to be a major route of infection for human immunodeficiency virus (HIV). Infection of human colon epithelial cells by HIV is not blocked by anti-CD4 antibodies known to block infection of lymphoid cells (J. Fantini, N. Yahi, and J. C. Chermann, Proc. Natl. Acad. Sci. USA 88:9297-9301, 1991), suggesting the presence of an alternate receptor for HIV on these cells. In this report, we show that (i) a monoclonal antibody specifically directed against galactosyl ceramide inhibited the infection of HT29 cells by two markedly different strains of HIV-1, as assessed by polymerase chain reaction amplification and reverse transcriptase assay; (ii) this antibody strongly labeled the surface of HT29 cells by immunofluorescence and electron microscopic immunolocalization; (iii) the labeling was preferentially but not totally restricted to the basolateral membrane domain of differentiated colonic cells, in agreement with the ability of HIV to infect both the apical and basolateral surfaces of these epithelial cells; and (iv) in thin-layer chromatography-immunostaining experiments with neutral glycolipids prepared from HT29 cells, the antibody specifically reacted with a ceramide monoglycoside fraction corresponding to galactosyl ceramide. We did not detect this glycolipid in lymphoid cells, and anti-galactosyl ceramide antibodies consistently failed to inhibit HIV infection of these cells. These data suggest that galactosyl ceramide (or a derivative) is an essential component of the receptor for HIV on the surface of HT29 cells.
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PMID:Galactosyl ceramide (or a closely related molecule) is the receptor for human immunodeficiency virus type 1 on human colon epithelial HT29 cells. 137 11

Phosphorothioate oligodeoxynucleotides exert a sequence-independent cytoprotective effect against human immunodeficiency virus type 1 (HIV-1). We now report that phosphorodithioate-containing oligodeoxycytidines are very potent inhibitors of HIV-1 reverse transcriptase in vitro, as they exhibit an increasing inhibitory effect with length and number of phosphorodithioate internucleotide linkages. This inhibitory effect can be at least 30-fold greater with phosphorodithioate oligodeoxycytidine than for the corresponding phosphorothioate analog of similar length. In cell culture, phosphorodithioate oligodeoxycytidines are active inhibitors of syncytia formation and effectively inhibit de novo infection of target cells by HIV-1. Moreover, comparative experiments show that a deoxycytidine phosphorodithioate 14-mer is as effective an inhibitor of de novo infection as a phosphorothioate-containing 28-mer. Such potent inhibition by oligomers of relatively short length makes dithioate analogs an additional class of potential therapeutic agents against acquired immunodeficiency syndrome.
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PMID:Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine. 137 23

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha [pJS3.7]. Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate [poly(rA).(dT)20] and polyribouridylate 20-mer of (3'-5')-deoxyadenylate [poly(rU).(dA)20] homopolymeric template-primers. Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates. Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively. The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured. Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT. In particular, poly(rA).(dT)20-[ddTMP] and poly(rU).(dA)20-[3'-dAMP] were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively. Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM. In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM. Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM. Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT. This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.
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PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers. 137 38

The relatively low fidelity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was implicated as a major factor that contributes to the genetic variability of the virus. Extension of mismatched 3' termini of the primer DNA was shown to be a major determinant of the infidelity of HIV-1 RT. Human immunodeficiency virus type 2 (HIV-2) also shows extensive genetic variations. Therefore, we have analyzed the fidelity of the DNA-dependent DNA polymerase activity of HIV-2 RT and compared it with those of RTs of HIV-1 and murine leukemia virus (MLV). Like other retroviral RTs, the HIV-2 RT was shown to lack a 3'----5' exonuclease activity. The ability of HIV-2 RT to extend preformed 3'-terminal A:A, A:C and A:G mispairs was examined by quantitating the amount and length of extended primers. The results demonstrate a relatively efficient mispair extension by HIV-2 RT with a specificity of A:C much greater than A:A greater than A:G. The mispair extension appears to be affected mainly by the increase of apparent Km values rather than by the change in Vmax values. The relative extension frequencies from all mispairs with HIV-1 and HIV-2 RTs was 6- to 9-fold greater than that of MLV RT, suggesting that the HIV enzymes are substantially more error-prone than MLV RT.
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PMID:Fidelity of the reverse transcriptase of human immunodeficiency virus type 2. 137 91

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein. The preparation of 23 murine monoclonal antibodies (mAb) against HIV-1 reverse transcriptase and their different effects on the enzyme are described. The interaction of purified mAbs with HIV-1 RT was demonstrated by enzyme-linked immunosorbent assay (ELISA), Western blots, and high performance liquid chromatography size exclusion chromatography. One of the antibodies also recognized recombinant HIV-2 RT. Antibody binding epitopes on HIV-1 RT were analyzed by immunoblotting using cyanogen bromide fragmented RT, C-terminally truncated mutants, and a peptide ELISA employing 15-mer synthetic overlapping peptides spanning nearly the complete polypeptide chain. The epitopes were mapped within three domains corresponding to amino acids 200-230, 300-428, and 528-560. Two mAbs show neutralizing properties on enzymatic functions of RT. One affects the polymerase activity and to a certain degree the RNase H activity of the enzyme, whereas the other inhibits the latter activity exclusively. mAb 28, which blocks the polymerase activity, interferes with the nucleotide binding region of RT, as shown by fluorescence spectroscopy using a labeled template/primer complex. By investigating the antibody effects on dimer formation of the heterodimeric enzyme, three domains corresponding to amino acids 230-300, 350-428, and residues around amino acid 540 involved in protein-protein interactions were localized.
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PMID:Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies. 137 37

We have examined the specificity of human immunodeficiency virus-1 (HIV-1) reverse transcriptase-associated RNase H in removing the tRNA(Lys3) (-)-strand primer in vitro using a model substrate. This substrate represents an intermediate in the reverse transcription process where the tRNA(Lys3) primer has not yet been removed after (+)-strand strong stop DNA synthesis. The substrate consists of an RNA oligonucleotide corresponding to the 3'-terminal 17 nucleotides of the tRNA(Lys3) linked to U5 DNA and annealed to single-stranded DNA containing the U5 and the primer-binding site. Upon incubation with HIV-1 reverse transcriptase p66/p51 heterodimer, the minus-strand DNA product resulting from RNase H cleavage retained the 3'-rA from the model tRNA primer. Changing the 3'-terminal AMP of the model tRNA primer from rA to dA did not alter the RNase H cleavage site. Further, the retention of AMP was not dependent on recognition of adjacent U5 sequences or the CCA terminus of the model tRNA(Lys3). The synthetic RNA primer was released as an intact species by a single endonucleolytic cleavage 5' of the rA. The cleavage patterns of Moloney murine leukemia virus and avian myoblastosis virus RNase H activities on the HIV-1 model substrate were more heterogeneous compared to HIV-1 RNase H. This specificity of HIV-1 RNase H would result in linear DNA molecules with a single rA at the U5 terminus and would provide two bases adjacent to the conserved CA dinucleotide to be cleaved away during the integration process.
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PMID:Specificity of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H in removal of the minus-strand primer, tRNA(Lys3). 137 44

The proposal that replication of human immunodeficiency virus type 1 (HIV-1), mediated by cell-to-cell transmission of the virus, might bypass de novo reverse transcription was tested by using one-step cell-to-cell and cell-free virus infection systems. Two well characterized reverse transcriptase (RT) inhibitors, azidothymidine at 20 microM and phosphonoformic acid at 100 micrograms/ml, blocked HIV replication completely following both cell-free virus and cell-to-cell transmission infection, as determined from the kinetics of unintegrated viral DNA synthesis and supernatant RT production after virus infection. Our results confirm that de novo reverse transcription is a crucial and mandatory event in HIV-1 replication following cell-to-cell transmission of the virus.
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PMID:De novo reverse transcription is a crucial event in cell-to-cell transmission of human immunodeficiency virus. 137 83

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.
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PMID:Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase. 137 42

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).
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PMID:Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase. 137 51

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