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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human immunodeficiency virus integrase (HIV IN) protein cleaves two nucleotides off the 3' end of viral DNA and subsequently integrates the viral DNA into target DNA. IN exposes a specific phosphodiester bond near the viral DNA end to nucleophilic attack by water or other nucleophiles, such as glycerol or the 3' hydroxyl group of the viral DNA molecule itself. Wild-type IN has a preference for water as the nucleophile; we here describe a class of IN mutants that preferentially use the 3' hydroxyl group of viral DNA as nucleophile. The amino acids that are altered in this class of mutants map near the putative active-site residues Asp-116 and Glu-152. These results support a model in which multiple amino acid side-chains are involved in presentation of the (soluble) nucleophile. IN is probably active as an oligomeric complex, in which the subunits have non-equivalent roles; we here report that nucleophile selection is determined by the subunit that supplies the active site.
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PMID:Identification of amino acids in HIV-2 integrase involved in site-specific hydrolysis and alcoholysis of viral DNA termini. 834 16

The integrase (IN) protein of human immunodeficiency virus type 1 (HIV-1) catalyzes site-specific cleavage of 2 bases from the viral long terminal repeat (LTR) sequence yet it binds DNA with little DNA sequence specificity. We have previously demonstrated that the C-terminal half of IN (amino acids 154-288) possesses a DNA binding domain. In order to further characterize this region, a series of clones expressing truncated forms of IN as N-terminal fusion proteins in E.coli were constructed and analyzed by Southwestern blotting. Proteins containing amino acids 1-263, 1-248 and 170-288 retained the ability to bind DNA, whereas a protein containing amino acids 1-180 showed no detectable DNA binding. This defines a DNA binding domain contained within amino acids 180-248. This region contains an arrangement of 9 lysine and arginine residues each separated by 2-4 amino acids (KxxxKxxxKxxxxRxxxRxxRxxxxKxxxKxxxK), spanning amino acids 211-244, which is conserved in all HIV-1 isolates. A clone expressing full-length IN with a C-terminal fusion of 16 amino acids was able to bind DNA comparably to a cloned protein with a free C-terminus, and an IN-specific monoclonal antibody which recognizes an epitope contained within amino acids 264-279 was unable to block DNA binding, supporting the evidence that a region necessary for binding lies upstream of amino acid 264.
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PMID:Characterization of a DNA binding domain in the C-terminus of HIV-1 integrase by deletion mutagenesis. 834 30

An obligatory step in retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-1 U5 LTR as substrate and supercoiled pSP65 DNA as target, we have investigated the effect of the polyanionic drug suramin on the catalytic activity of the IN protein. It was found that at stoichiometric suramin to protein ratios, suramin displays a strong inhibitory effect on both the processing and strand transfer reactions. This inhibitory effect is related to the decrease of IN protein binding efficiency to the LTR end DNA fragment.
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PMID:Inhibitory effect of the polyanionic drug suramin on the in vitro HIV DNA integration reaction. 837

Retroviral growth requires as an obligatory step the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the LTR ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of the HIV-I U5 LTR as substrate and supercoiled pSP65 DNA as target, we have measured the effect of various topoisomerase inhibitors on the functional activity of the IN protein. Among the various drugs tested, the antitumor drug 2N-Methyl, 9-hydroxyellipticinium (NMHE) displays a marked inhibitory effect on the IN-catalyzed U5 insertion. This effect is related to the DNA binding properties of the drug rather than to a selective effect on the IN protein or the DNA-IN protein complex.
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PMID:Effect of topoisomerase inhibitors on the in vitro HIV DNA integration reaction. 838 50

The preintegration complex of human immunodeficiency virus type 1 (HIV-1) is a large nucleoprotein complex containing viral nucleic acids in association with products of the viral gag and pol genes. One of these proteins, integrase, is absolutely required for the integration and formation of the provirus. Although HIV-1-specific 2-LTR circles from nuclei of HIV-1-infected cells were found to be associated within a high-molecular-weight nucleoprotein complex, antibodies to HIV-1 integrase failed to precipitate this form of viral DNA. This result indicates that circular forms of HIV-1 DNA are not associated with integrase. These viral DNA forms seem to exist in a context of a nucleoprotein complex that is different from a preintegration complex of HIV-1.
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PMID:Human immunodeficiency virus type 1 2-LTR circles reside in a nucleoprotein complex which is different from the preintegration complex. 841 90

The retroviral integrase (IN) protein is essential for integration of retroviral DNA into the host cell genome. To identify functional domains within the protein and to assess the importance of conserved residues, we performed site-directed mutagenesis of HIV-1 IN and analyzed the mutants in vitro for IN-mediated activities: 3' processing (att site-specific nuclease activity), strand transfer (the joining of att site oligonucleotides to target DNA), disintegration (the reverse of strand transfer), and integration site selection. Changing the conserved residue His-16 either to Cys or to Val in a proposed zinc-finger region had minimal effect on IN activities. Alteration of two highly conserved amino acid residues, Asp-116-->Ile and Glu-152-->Gly, each resulted in complete or nearly complete loss of 3' processing, strand transfer, and disintegration, whereas alteration of another conserved residue, Trp-235-->Glu, had no demonstrable effect on any of the activities in vitro. Two mutants, Asp-64-->Val and Arg-199-->Cys delta, each demonstrated differential effects on IN activities. Asp-64-->Val has no demonstrable strand transfer or disintegration activity yet maintains 3' processing activity at a diminished level. Arg-199-->Cys delta, which lacks part of the carboxyl terminus of IN, has impaired strand transfer activity without loss of disintegration activity. Use of a target site selection assay showed that all of our mutants with strand transfer activity maintain the same integration pattern as wild type IN. We conclude that not all highly conserved IN residues are essential for IN activities in vitro, zinc coordination by the proposed zinc-finger domain may not be required for the activities assayed, alteration of single residues can yield differential effects on IN activities, and target site selection into naked DNA is not necessarily altered by changes in strand transfer activity.
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PMID:Site-directed mutagenesis of HIV-1 integrase demonstrates differential effects on integrase functions in vitro. 842 Sep 82

An obligatory step of retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host. Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the long terminal repeat (LTR) ends and the strand transfer reaction. Using the 3' end of synthetic oligonucleotides which match the termini of HIV-1 LTRs as substrate and supercoiled pSP65 DNA as the target, we describe an assay that is suitable for the enzymatic analysis of the integration and for testing candidate inhibitors of HIV IN protein.
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PMID:Quantitative in vitro assay for human immunodeficiency virus deoxyribonucleic acid integration. 843 53

The expression of the pol gene of human immunodeficiency virus type 1 (HIV-1) occurs by a ribosomal frameshift between the gag and the pol genes. The Gag-Pol polyprotein is produced at levels of 5 to 10% of that of the Gag protein, and is incorporated into virions to provide the viral protease, reverse transcriptase, and integrase which are essential for replication. The mechanism(s) by which the Gag-Pol polyprotein are targeted to the HIV virion is unknown, although it is believed to be via an interaction with the Gag protein. To further explore the mechanism by which the Gag-Pol polyprotein is incorporated into virions, we have constructed a mutation which changes an aspartic acid in the protease active site to asparagine (pHXB2pro-); a four-amino-acid insertion into the protease gene (pHXB2Smal); and insertion of translational termination codons in the protease gene following the gag gene (pHXB55). Transfection of these proviral genomes into COS-1 cells resulted in intracellular expression of only Pr55gag, demonstrating the inactivation of the viral protease. The expression of Pr55gag was evident in cells transfected with pHXB2pro- during a short pulse and first 3 hr of chase period, whereas at later times the intracellular levels of Pr55gag were greatly reduced. In contrast, the intracellular Pr55gag expressed from transfection of pHXB2Smal or pHXB55 were evident even after 6- or 12-hr chase times. To ascertain the effects of the mutations on the assembly and release of viruslike particles, the supernatants from the transfected cells were analyzed for the presence of Pr55gag. The release of Pr55gag from cells transfected with pHXB2pro- occurred as early as 1 hr following chase period, and increased for up to 3 hr. In contrast, reduced levels of Pr55gag were detected in the medium from cells transfected with pHXB2Smal or pHXB55. Subcellular fractionation studies demonstrated that the Pr55gag expressed from transfection of pHXB2pro- was rapidly targeted to intracellular membranes, while the majority of the Pr55gag expressed from transfection of pHXB2Smal or pHXB55 was distributed evenly between the cytoplasm and membrane fractions. Finally, the released viruslike particles obtained from the transfection of proviral genome pHXB2pro- were stable to mild detergent treatment, whereas particles obtained from transfection of pHXB2Smal and pHXB55 were relatively unstable. These results demonstrate that subtle changes in the Gag-Pol polyprotein of HIV-1 can have significant effects on the assembly and physical stability of the released virus.
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PMID:Mutations in the protease gene of human immunodeficiency virus type 1 affect release and stability of virus particles. 850 89

We have previously identified a residue in the carboxyl terminus of human immunodeficiency virus type 1 integrase (HIV-1 IN), W-235, the requirement for which is only revealed in viral assays for integrase function (P. M. Cannon, W. Wilson, E. Byles, S. M. Kingsman, and A. J. Kingsman, J. Virol. 68:4768-4775, 1994). Our further analysis of this region of retroviral IN has now identified several sequence motifs which are conserved in all the retroviruses we examined, apart from human spumaretrovirus. We have made mutations within these motifs in HIV-1 IN and examined their phenotypes when reintroduced into an infectious proviral clone. The deleterious effects of several of these mutations demonstrate the importance of these regions for IN function in vivo. We observed a further discrepancy, at a motif that is only conserved in the lentiviruses, in the ability of mutants to function in in vitro and in vivo assays. Substitutions both in this region and at W-235 abolish HIV-1 infectivity but do not affect particle production, morphology, reverse transcription, or nuclear import in T-cell lines. Taken together with the in vitro data suggesting that neither of these residues is directly involved in the catalytic reactions of IN, it seems likely that we have identified regions of IN that are essential for interactions with other components of the integration machinery.
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PMID:Conserved sequences in the carboxyl terminus of integrase that are essential for human immunodeficiency virus type 1 replication. 852 88

A mammalian expression vector designed for production of HIV-1 integrase was found to enhance the stability of a linear reporter plasmid in COS-7 cells. The effect is strictly dependent on coexpression of the HIV-1 rev gene and on the inclusion of U3 and U5 portions of the HIV-1 LTR in the reporter plasmid. Integrase point mutations P109S and D116N drastically reduced stabilization whereas T115A and D64A had little or no effect. Immunoblot analysis revealed the presence of a 32-34kDa integrase protein in extracts of transfected COS-7 cells and of wild type and mutant integrase proteins at comparable levels. We conclude that integrase acts in trans in COS-7 cells, possibly by binding to the HIV-1 LTR in the plasmid. This transfection system may be useful for studying factors that stabilize the HIV-1 DNA genome prior to its integration into the host cell chromosome.
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PMID:Human immunodeficiency virus type 1 integrase stabilizes a linearized HIV-1 LTR plasmid in vivo. 852 37


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