Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We were able to isolate high-affinity RNAs from a random pool that binds to integrase protein from the human immunodeficiency virus-type 1 using the procedure now known as SELEX. Generally, the RNAs fell into three different classes in binding buffer containing 250 mM NaCl: group I class of molecules binds integrase with a dissociation constant (Kd) on the order of 10 nM, group II molecules had a Kd of about 80 nM, and group III about 800 nM. The RNA with the highest affinity from the group I class of molecules, designated P5, was characterized using computer modeling, chemical and enzymatic probing, and deletion analysis. Our secondary structure model for this RNA suggests interactions between looped-out fixed nucleotides and nucleotides from the randomized region; a GNRA tetraloop is also in the structure. We showed that our integrase was able to process a U5 mimic in vitro. P5 competes effectively for binding with the double-stranded DNA mimic of U5 at 180 mM NaCl concentration.
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PMID:Isolation of high-affinity RNA ligands to HIV-1 integrase from a random pool. 777 67

Upon entry into a host cell, retroviruses direct the reverse transcription of the viral RNA genome and the establishment of an integrated proviral DNA. The retroviral integrase protein (IN) is responsible for the insertion of the viral DNA into host chromosomal targets. The two-hybrid system was used to identify a human gene product that binds tightly to the human immunodeficiency virus-type 1 (HIV-1) integrase in vitro and stimulates its DNA-joining activity. The sequence of the gene suggests that the protein is a human homolog of yeast SNF5, a transcriptional activator required for high-level expression of many genes. The gene, termed INI1 (for integrase interactor 1), may encode a nuclear factor that promotes integration and targets incoming viral DNA to active genes.
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PMID:Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5. 780 Nov 19

The efficiency of detection of 2 sets of primer pairs from putatively conserved regions of the human immunodeficiency virus type 2 (HIV-2) genome were assessed in 86 seropositive individuals from The Gambia by nested polymerase chain reaction (PCR). HIV-2 long terminal repeat (LTR) target sequences were detected in DNA extracted from peripheral blood mononuclear cells (PBMCs) in 84 of 86 (97%) individuals whereas HIV-2 integrase (pol) gene sequences were detected in 39 of 41 (95%) individuals. The use of LTR target sequences and recombinant Pfu DNA polymerase, rather than Taq polymerase, in a modified secondary amplification reaction mediated the incorporation of 35S-labeled nucleotides in a quantitative radiometric assay. This sensitive assay was used to quantify HIV-2 proviral DNA in clinical samples and compared well with estimations by limiting end-point dilution of target molecules. A linear response between counts and the number of copies amplified from serial dilutions of pROD10 plasmid DNA (3-2000 copies) yielded a standard curve to allow extrapolation to clinical data. Increased levels of HIV-2 proviral DNA, expressed as copies per 10(5) CD4-positive lymphocytes, were associated with declining CD4 count in 63 adult patients (Spearman rank correlation, r = -0.71, n = 63, p < 0.001) and with the occurrence of HIV-related clinical disease. Kruskall-Wallis analysis of variance analysis showed the mean proviral copy number (log10) to be significantly different between groups (p < 0.001) where CD4 counts were grouped as < 200/mm3 (3.4 +/- 1.05 copies), 200-500/mm3 (2.84 +/- 0.93 copies), and > 500/mm3 (1.88 +/- 0.43 copies).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:HIV type 2 proviral load measured by quantitative polymerase chain reaction correlates with CD4+ lymphopenia in HIV type 2-infected individuals. 781 34

SDZ NIM 811 is a cyclosporin A analog that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. The mechanism of action of SDZ NIM 811 is clearly different from those of all other anti-HIV agents described so far. In cell-free assays, it is not an inhibitor of reverse transcriptase, protease, integrase, and it does not interfere with Rev or Tat function. SDZ NIM 811 does not down-regulate CD4 or inhibit fusion between infected and uninfected, CD4-expressing cells. p24 production from chronically HIV-infected cells is not impaired either. To elucidate the mode of action of SDZ NIM 811, we performed DNA PCR analysis in HIV-1 IIIB-infected MT4 cells in one cycle of virus replication. The effects of SDZ NIM 811 on the kinetics of viral DNA synthesis, appearance of two-long terminal repeat circles (2-LTR circles), and integration of DNA were studied. SDZ NIM 811 inhibited 2-LTR circle formation in a concentration-dependent manner, which is indicative of nuclear localization of preintegration complexes. Half-maximal inhibition was achieved at 0.17 microgram/ml; this concentration is close to the 50% inhibitory concentrations (0.01 to 0.2 microgram/ml) for viral growth inhibition. As expected, integration of proviral DNA into cellular DNA was also inhibited by SDZ NIM 811. Analysis of the viral particles produced by SDZ NIM 811-treated, chronically infected cells revealed amounts of capsid proteins, reverse transcriptase activity, and viral RNA comparable to those of the untreated control. However, these particles showed a dose-dependent reduction in infectivity (50% inhibitory concentration of 0.028 microgram/ml) which indicates that the assembly process is also impaired by SDZ NIM 811. Gag proteins are postulated to play a role not only in assembly but also in early steps of viral replication, e.g., nuclear localization of the preintegration complex. Recently, it was reported that HIV-1 Gag protein binds to cyclophilin A, the intracellular receptor for cyclosporin A. Interference with Gag-cyclophilin interaction may be the molecular basis for the antiviral activity of cyclosporin A and its analogs.
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PMID:Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus type 1 (HIV-1): interference with early and late events in HIV-1 replication. 781 48

The retroviral integrase (IN) is a virus-encoded enzyme that is essential for insertion of viral DNA into the host chromosome. In order to map and define the properties of a minimal functional domain for this unique viral enzyme, a series of N- and C-terminal deletions of both Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate representing the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Though deficient for processing, the RSV deletion mutants exhibited a secondary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage-ligation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a minimal functional central region of approximately 140 amino acids for each enzyme that includes the highly conserved D,D(35)E domain. Results with HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage-ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even the isolated central region of HIV-1 IN exhibited this sequence requirement for optimal activity. We conclude that this evolutionarily conserved central region of IN not only encodes residues that are required for the catalytic activity of the enzyme but also harbors some or all of the determinants responsible for recognition of the CA/GT dinucleotides that are present at the ends of all retroviral DNAs.
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PMID:Activities and substrate specificity of the evolutionarily conserved central domain of retroviral integrase. 783

Ribozymes offer a potentially important way to inactivate intracellular RNA from almost any gene whose nucleotide sequence is known. Recently, we found that hammerhead ribozymes directed against mRNA of tumour necrosis factor alpha (TNF alpha) and its derivatives, preferentially bind to a cellular protein(s). To better understand the effect of different 3'-terminal hairpins on ribozyme stability as well as their effect on the protein binding to the ribozyme, a mathematical treatment of the decay of three TNF alpha ribozymes that differed at their 3' ends was performed. One ribozyme contained a 3'-terminal hairpin derived from a transcription terminator of bacteriophage T7, another contained the same hairpin but modified to be highly enriched for G+C nucleotides, and a third lacked a hairpin. The TNF alpha ribozyme decay had two kinetic components. The slow component exhibited exponential decay with a half life of approximately 250 h in all cases. The 3'-terminal hairpin has no significant effect on this component. This slow phase accounted for 60-80% of ribozyme decay. The rapid phase also exhibited exponential decay. For this phase, a 3'-terminal hairpin roughly doubled the half-life (1.7-3.4). The slow phase of degradation was about three times faster for a ribozyme directed at the integrase mRNA of human immunodeficiency virus-1 than that seen with the TNF alpha ribozyme. Taken together, these results suggest that the ribozyme population is initially sensitive to degradation, with the presence of a hairpin provides some protection, and indicate that the addition of the hairpin to the ribozyme did not prevent the in vivo additional stabilizing effect of the protein(s).
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PMID:In vivo decay kinetic parameters of hammerhead ribozymes. 783 9

The human spuma retrovirus or foamy virus integrase (HFV IN) is an enzymatically active protein consisting of domains similar to other retroviral integrases: an amino-terminal HH-CC finger, a centrally located region with the conserved D, D-35-E protein motif required for catalytic activity and oligomerization, and at least one DNA binding domain implicated in the 3' DNA processing activity and integrase. Recombinant, purified HFV IN protein carrying 10 histidine residues displays a site-specific endonuclease, an integrase, and a disintegrase activity with oligonucleotide substrates that mimic the viral long terminal repeat (LTR) ends. Site-directed mutagenesis of conserved HFV IN residues of the catalytic domain had increased endonuclease and disintegrase activities. Deletion mutants at both ends of the HFV IN protein were generated, purified, and characterized. Unexpectedly, it was found that the HFV integrase and disintegrase activities require an intact NH2-terminal sequence and that COOH-terminal deletions led to an increase in disintegrase activity. The HH-CC finger of HFV IN was exchanged with that of the human immunodeficiency virus-1 (HIV-1) IN protein. The resulting chimeric IN had a 3' processing activity that utilized the HFV LTR instead of the HIV LTR, indicating that the central domain is crucial for substrate recognition. Functional complementation of the amino-terminal deletion mutant of HFV IN was achieved by a carboxyl-terminal deletion mutant of the chimeric IN, resulting in high levels of integrase activity.
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PMID:Characterization of the human spuma retrovirus integrase by site-directed mutagenesis, by complementation analysis, and by swapping the zinc finger domain of HIV-1. 785 75

Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved "DD35E" motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.
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PMID:An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer. 785 18

DNA copies of the human immunodeficiency virus (HIV) genome integrate nonrandomly into the chromosomal DNA of the host cell. In this report, we investigate the molecular basis of this selectivity using the virus-encoded HIV integrase to direct integration of a synthetic HIV long terminal repeat substrate into either DNA molecules of known structure or previously defined nucleosomal complexes. We find that the structure of the target greatly influences the site of integration, and, moreover, DNA curvature, flexibility, and rigidity in solution all influence the frequency of integration. Importantly, for DNA with all of these properties, the distortion of the double helix directed by association with the histone proteins promotes the integration reaction and alters the distribution of sites that are selected for integration. We suggest that both intrinsic DNA structure and the folding of DNA into chromosomal structures will exert a major influence on target site selection for integration of the viral genome.
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PMID:The influence of DNA and nucleosome structure on integration events directed by HIV integrase. 792 89

The integrase (IN) protein of the human immunodeficiency virus (HIV) mediates two distinct reactions: (i) specific removal of two nucleotides from the 3' ends of the viral DNA and (ii) integration of the viral DNA into target DNA. Although IN discriminates between specific (viral) DNA and nonspecific DNA in physical in vitro assays, a sequence-specific DNA-binding domain could not be identified in the protein. A nonspecific DNA-binding domain, however, was found at the C terminus of the protein. We examined the DNA-binding characteristics of HIV-1 IN, and found that a stable complex of IN and viral DNA is formed in the presence of Mn2+. The IN-viral DNA complex is resistant to challenge by an excess of competitor DNA. Stable binding of IN to the viral DNA requires that the protein contains an intact N-terminal domain and active site (in the central region of the protein), in addition to the C-terminal DNA-binding domain.
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PMID:Formation of a stable complex between the human immunodeficiency virus integrase protein and viral DNA. 793 34


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