Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for the rapid preparation of a defined substrate to monitor RNase H activity has been developed. Using this substrate, we have investigated the RNase H activities of the different forms of recombinant HIV-1 and HIV-2 reverse transcriptase (RT) in detail. As we report here, RNase H activity is associated only with the dimeric forms (p51/p66 or p66/p66) of the enzymes.
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PMID:RNase H activity of HIV reverse transcriptases is confined exclusively to the dimeric forms. 137 72

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.
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PMID:Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities. 137 41

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280. In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis. Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified. In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme. The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested. The double cysteine mutant RT, on the other hand, exhibits several unique features. First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes. This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar. Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT. The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs. Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs. The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins.
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PMID:The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1. 137 33

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 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

L-696,229 (3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2 (1H)-one) is a specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity that possesses antiviral activity in cell culture (W.S. Saari, J.M. Hoffman, J.S. Wai, T.E. Fisher, C.S. Rooney, A.M. Smith, C.M. Thomas, M. E. Goldman, J. A. O'Brien, J. H. Nunberg, J. C. Quintero, W. A. Schleif, E. A. Emini, and P. S. Anderson, J. Med. Chem. 34:2922-2925, 1991). In the present study, the RT-inhibitory activity and antiviral properties were characterized in detail. The inhibition of RT activity was template-primer dependent with 50% inhibitory concentrations of 0.018 to 0.50 microM and was noncompetitive with respect to deoxynucleoside triphosphates. L-696,229 inhibited RT activity in a mutually exclusive manner with respect to either phosphonoformate or azidothymidine triphosphate and was a weak partial inhibitor of the RNase H activity associated with HIV-1 RT. The compound did not significantly inhibit other retroviral or cellular polymerases at 300 microM.L-696,229 inhibited the spread of HIV-1 infection in cell cultures with all cell types and viral isolates tested, including human peripheral blood mononuclear cells and a virus isolate resistant to azidothymidine.
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PMID:L-696,229 specifically inhibits human immunodeficiency virus type 1 reverse transcriptase and possesses antiviral activity in vitro. 138 Jul 88

We have investigated the ability of pyridoxal-5'-phosphate to inhibit a recombinant deletion mutant of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) which is missing the last 23 amino acids of the C-terminus. This mutant reverse transcriptase is characterized by normal polymerase activity as compared with full-length enzyme; however, it has no RNase H activity. Inhibition studies with pyridoxal-5'-phosphate showed several differences as compared with inhibition of full-length enzyme: (1) Inhibition of mutant reverse transcriptase was independent of divalent cation, (2) Either substrate alone could protect mutant reverse transcriptase from inactivation by pyridoxal-5'-phosphate, and (3) stoichiometry of pyridoxal-5'-phosphate binding to mutant reverse transcriptase was 2 mol/mol under the same conditions in which 1 mol/mol bound to full-length enzyme. Furthermore, in the presence of either substrate alone, the stoichiometry of pyridoxal-5'-phosphate binding to the mutant was reduced to 1 mol/mol. These results indicate that the second binding site for pyridoxal-5'-phosphate seen in the mutant reverse transcriptase is at or near the primer-template binding site of the enzyme. They also suggest that the RNase H domain of HIV RT plays a functional role in substrate binding at the polymerase domain.
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PMID:Pyridoxal-5'-phosphate inhibits the polymerase activity of a recombinant RNAase H-deficient mutant of HIV-1 reverse transcriptase. 138 Dec 4

In a search for compounds active against human immunodeficiency virus type 1 (HIV-1), it was found that the novel low-molecular weight immunoenhancer ammonium trichloro(dioxyethylene-O,O'-) tellurate (AS101) suppresses production of HIV-1 in vitro. Treatment of HIV-1-infected peripheral blood mononuclear cells (PBMC) with increasing concentrations of AS101 resulted in substantial inhibition of virus production as measured by both reverse transcriptase (RT) activity and antigen presence in supernatants of treated cells. AS101 had no effect on PBMC viability, growth, or morphology up to a concentration of 15 microM for 14 days. To elucidate a possible mechanism for the inhibition of AS101, we have analyzed the effect of the drug on the catalytic functions associated with HIV RT, namely the RDDP, DDDP, and RNase H activities. RDDP and DDDP activities were impaired by the drug with calculated IC50 value of about 4 microM. On the other hand, the RNase H activity was less sensitive to AS101, with an apparent IC50 value of about 30 microM. The anti-HIV-1 activity of AS101 as reflected by inhibition of the different catalytic functions associated with viral RT, in the absence of drug-related toxicity to lymphocytes, together with its immunomodulating activity strongly argues in favor of its evaluation, as a therapeutic agent for patients with HIV infection.
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PMID:Inhibition of the reverse transcriptase activity and replication of human immunodeficiency virus type 1 by AS 101 in vitro. 138 Dec 5

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52


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