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: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed two human immunodeficiency virus (HIV-1) reverse transcriptase mutants of helix H in the thumb subdomain suggested by x-ray crystallography to interact with the primer strand of the template-primer. These enzymes, G262A and W266A, were previously shown to have greatly elevated dissociation rate constants for template-primer and to be much less sensitive to inhibition by 3'-azidodeoxythymidine 5'-triphosphate. Here we describe their processivity and error specificity. The results reveal that: (i) both enzymes have reduced processivity and lower fidelity for template-primer slippage errors, (ii) they differ from each other in sequence-dependent termination of processive synthesis and in error specificity, and (iii) the magnitude of the mutator effect relative to wild-type enzyme for deletions in homopolymeric sequences decreases as the length of the run increases. Thus amino acid substitutions in a subdomain thought to interact with the duplex template-primer confer a strand slippage mutator phenotype to a replicative DNA polymerase. This suggests that interactions between specific amino acids and the primer stem at positions well removed from the active site are critical determinants of processivity and fidelity. These effects, obtained in aqueous solution during catalytic cycling, are consistent with and support the existing crystallographic structural model.
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PMID:Reduced frameshift fidelity and processivity of HIV-1 reverse transcriptase mutants containing alanine substitutions in helix H of the thumb subdomain. 754

L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.
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PMID:Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate. 754 86

Foscarnet is a broad-spectrum viral DNA polymerase inhibitor active in vitro and in vivo against human immunodeficiency virus type 1 (HIV-1). Strains of HIV-1 resistant to foscarnet were selected by in vitro passage in increasing concentrations of drug. Reduced susceptibility to foscarnet was evident at the levels of both HIV-1 replication and reverse transcriptase. Biologically cloned, foscarnet-resistant strains with distinct genotypes were hypersensitive to zidovudine, azidodeoxyuridine, nevirapine, and R82913 but had unchanged susceptibility to zalcitibine and didanosine. The reverse transcriptase of foscarnet-resistant strains had unique substitutions Glu89-Lys, Leu92-Ile, or Ser156-Ala, the third being associated with six polymorphic changes. Introduction of these mutations into wild-type HIV-1 by site-directed mutagenesis confirmed their role in foscarnet resistance. In the three-dimensional structure of the reverse transcriptase enzyme these amino acids are located close to the template strand of the template primer and far away from the putative pyrophosphate binding site, suggesting that the mechanism by which HIV-1 becomes resistant to foscarnet is indirect. Foscarnet resistance is thus likely to be mediated through an altered interaction of the mutant enzyme with the template strand of the template primer which distorts the geometry of the polymerase active site and thereby decreases foscarnet binding.
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PMID:Characterisation of foscarnet-resistant strains of human immunodeficiency virus type 1. 754 54

We have previously noted an association between proviral load and the severity of immune disease in individuals with a wide range of CD4 cell counts. Using the quantitative DNA polymerase chain reaction technology developed in our laboratory, we sought to extend these observations, with a view to establishing guidelines for the use of proviral load in a clinical context. We studied 199 patients with a range of CD4 cell counts attending an urban tertiary care center. Proviral load/10(6) peripheral blood mononuclear cells (PBMCs) was measured using a microtiter plate assay designed specifically for this purpose. Human immunodeficiency virus proviral DNA was detected in 193 of 199 clinical samples. Levels of proviral load were tabulated for patients and evaluated in seven categories defined by CD4 cell counts. Although a wide range of proviral loads was observed in each category of patients, there was a trend toward increasing proviral load with decreasing CD4 cell count. Statistically significant relationships were observed between proviral load and the CD4 cell count and the CD4 cell percentage (Spearman's correlation coefficient -0.19, p = 0.01 for both absolute CD4 and CD4 percentage). These relationships were quite weak and could not be taken to explain disease progression in isolation. If we defined a cutoff between low and high proviral loads at 100 copies/10(6) PBMCs, we noted that 52% (24 of 46) of patients with CD4 cell counts > 400/microliters had lower loads, as compared with 16% (24 of 143) of those with more advanced disease (p < 0.01). There is a weak, but statistically significant association between proviral load and CD4 cell depletion.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Potential applications of proviral load measurement in clinical retrovirology. 755 12

Swertifrancheside [1], a new flavonone-xanthone glucoside isolated from Swertia franchetiana, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2], a triterpene isolated from the roots of Maprounea africana, and protolichesterinic acid [3], an aliphatic alpha-methylene-gamma-lactone isolated from the lichen Cetraria islandica, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT), with 50% inhibitory doses (IC50 values) of 43, 3.7, and 24 microM, respectively. They were not cytotoxic with cultured mammalian cells. The kinetic mechanisms by which compounds 1-3 inhibited HIV-1 RT were studied as was their potential to inhibit other nucleic acid polymerases. Swertifrancheside [1] bound to DNA and was shown to be a competitive inhibitor with respect to template-primer, but a mixed-type competitive inhibitor with respect to TTP. On the other hand, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] were mixed-type competitive inhibitors with respect to template-primer and noncompetitive inhibitors with respect to TTP. Therefore, the mechanism of action of 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] as HIV-1 RT inhibitors involves nonspecific binding to the enzyme at nonsubstrate binding sites, whereas swertifrancheside [1] inhibits enzyme activity by binding to the template-primer.
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PMID:Mechanistic evaluation of new plant-derived compounds that inhibit HIV-1 reverse transcriptase. 756 95

A 3'-->5' exonuclease has been highly purified from the cytosol of human acute lymphoblastic leukemia H9 cells. The apparent molecular weight of this enzyme was approximately 50,000, as indicated by its sedimentation in glycerol gradients. The exonuclease did not copurify with DNA polymerase activity, required MgCl2 for its exonucleolytic activity, and was inhibited by KCl above 60 mM. The enzyme was active on single-stranded DNA, DNA duplexes and DNA/RNA duplexes, and it was efficient at removing 3'-terminal mispairs from DNA. The products of the exonucleolytic reaction were deoxynucleoside 5'-monophosphates. The behavior of the exonuclease was examined on DNA terminated at the 3' end with a variety of dideoxynucleosides that are potent against human immunodeficiency virus type 1. The exonuclease has a broad substrate specificity; however, the rate of the enzymatic reaction varied among the D dideoxynucleosides tested (ddAMP = ddCMP > d4TMP > AZTMP). Similarly, the enzyme was examined for its reactivity with DNA terminated by either the D or L enantiomers of ddC, SddC or FddC. The removal of analogs with the native D configuration was at least 6-fold more rapid than that of the L-compounds, and the type of structural modification had an impact on the rate at which the D enantiomers were removed (SddCMP > ddCMP > FddCMP). The monophosphate forms of AZT, D4T, L-FddC and L-ddC were potent inhibitors of the exonuclease at micromolar concentrations, while D-ddCMP partially inhibited the enzyme at millimolar concentrations. Based on its physical and enzymatic properties, this exonuclease represents a novel enzyme that may have an important role in determining the relative potencies of dideoxynucleosides against human immunodeficiency virus type 1.
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PMID:Removal of anti-human immunodeficiency virus 2',3'-dideoxynucleoside monophosphates from DNA by a novel human cytosolic 3'-->5' exonuclease. 757 43

We have studied the effects of four nonnucleoside inhibitors, including the novel natural product inhibitor calanolide A, on molecular chimeras containing complementary segments of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs). All four compounds specifically inhibited the DNA polymerase activity of HIV-1 RT but had no apparent effect on the RNase H activity of this enzyme or on the DNA polymerase or RNase H activity of HIV-2 RT. Three of these compounds showed the generally expected patterns of resistance and susceptibility with the various chimeric RTs. However, the inhibition patterns of the chimeric RTs by calanolide A provided evidence that there is a segment between residues 94 and 157 in HIV-1 RT that is critical for inhibition. However, the data also suggest that there may be a second segment located between amino acids 225 and 427 in HIV-1 RT that is also important for specifying susceptibility to the drug.
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PMID:Specific inhibition of the reverse transcriptase of human immunodeficiency virus type 1 and the chimeric enzymes of human immunodeficiency virus type 1 and type 2 by nonnucleoside inhibitors. 768 94

5'-Triphosphates of 1-(2',3'-epithio-2',3'-dideoxy-beta-D- lyxofuranosyl)thymine, 1-(2',3'-epithio-2',3'-dideoxy-beta-D-ribofuranosyl)thymine and 2',3'-lyxoanhydrothymidine have been shown to be termination substrates for human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) reverse transcriptases as well as DNA polymerase I from E. coli and DNA polymerase beta from rat liver. At the same time they do not terminate DNA synthesis catalysed by DNA polymerase epsilon from human placenta. Km values of ltTTP, rtTTP and laTTP incorporation into the DNA chain during catalysis by AMV reverse transcriptase agree closely with each other being 1.5-2.5 times higher than Km value for dTTP. Furthermore, Vmax values for modified substrates are only 2-3 times lower than Vmax for dTTP. The evidence favours the hypothesis of high affinity of modified nucleotides with a flattened furanosyl ring for DNA polymerase active sites.
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PMID:Modified nucleoside 5'-triphosphates containing 2',3'-fused three-membered rings as substrates for different DNA polymerases. 768 65

We have successfully expressed and purified the human immunodeficiency virus type-1 reverse transcriptase (RT) using the baculovirus expression vector system. This expression system provides a eukaryotic environment in which post-translational modifications of foreign gene products can occur. After infection with recombinant virus, Western blot analysis confirmed the presence of an immunoreactive polypeptide of approximately 66 kDa from insect Sf9 cell lysates. RT was then purified from crude extracts of baculovirus-infected Sf9 cells; SDS-PAGE analysis of fractions obtain from partial purification showed that in contrast to the Escherichia coli-expressed RT, the baculovirus-expressed RT corresponded to a doublet of peptides at approximately 66 kDa. Further purification of the protein resulted in a p66 protein, judged to be more than 90% pure by SDS-PAGE and Coomassie blue stain. Following purification, the baculovirus derived RT had specific activity for DNA polymerase similar to that of the E. coli-derived RT. Therefore, RT purified from Sf9 cells appears to be suitable for structure-function studies of this enzyme.
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PMID:Expression and purification of the HIV-1 reverse transcriptase using the baculovirus expression vector system. 769 Jun 27

The reverse transcriptase of equine infectious anemia virus (EIAV) shows sequence similarity with the reverse transcriptases of other lentiviruses, particularly with those of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2). We have constructed a plasmid that when introduced into E. coli induces the synthesis of substantial quantities of the nearly authentic EIAV reverse transcriptase. The viral and bacterially expressed reverse transcriptases are similar in their molecular weights. The bacterial expression clone was used to generate deletion mutants of the protein. Mutations in both amino and carboxyl terminal regions of the polypeptide strongly affect the DNA polymerase activity of the enzyme. Thus, EIAV reverse transcriptase resembles the reverse transcriptases of HIV-1 and HIV-2 and can serve as a suitable enzyme for studying the structure-function relationship in lentiviral reverse transcriptase.
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PMID:Expression and mutational analysis of the reverse transcriptase of the lentivirus equine infectious anemia virus. 769 81


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