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)

Azodicarbonamide tested as an anti-HIV agent was reported to expulse zinc from viral zinc-cysteine factors and to inhibit calcium mobilization machinery. It has structural analogy with hydroxyurea that inhibits ribonucleotide reductase and could also act on this target. Azodicarbonamide was therefore tested for its capacity to modulate deoxyribonucleotides triphosphate pools alone or in combination with other agents in the lymphoblastic SUP-T1 cell line susceptible to HIV infection. The deoxyribonucleotides triphosphate were evaluated by an enzymatic assay using sequenase. Two hours exposure of SUP-T1 cells to 100 microM azodicarbonamide induced a 50% reduction of each deoxyribonucleotide triphosphate. Among other inhibitors of nucleotide metabolism (hydroxyurea, methotrexate and thymidine), hydroxyurea only reproduces the effect of azodicarbonamide. This suggests, but does not demonstrate directly, that azodicarbonamide inhibits ribonucleotide reductase activity. The combination of azodicarbonamide with each of these inhibitors affected particularly the dCTP pool. During this study it was also suggested that azodicarbonamide could interfere with thymidine phosphorylation. Thymidine phosphorylating activity was measured with 3H-thymidine as substrate. In acellular preparations, azodicarbonamide also non-competitively inhibits thymidine phosphorylating activity. This effect was not reproduced by hydroxyurea. Thus, in vitro azodicarbonamide decreases the intracellular pool of deoxyribonucleotide and thymidine phosphorylation.
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PMID:Ribonucleotide reductase and thymidine phosphorylation: two potential targets of azodicarbonamide. 1214 96

Human monocytes/macrophages are target cells for HIV-1 infection. As other non-dividing cells, they are characterized by low and imbalanced intracellular dNTP pool levels and an excess of dUTP. The replication of HIV-1 in this cellular context favors misincorporation of uracil residues into viral DNA because of the use of dUTP in place of dCTP. We have previously reported that the host uracil DNA glycosylase enzyme UNG2 is packaged into HIV-1 viral particles via a specific association with the integrase domain of the Gag-Pol precursor. In this study, we investigated whether virion-associated UNG2 plays a role similar to that of its cellular counterpart. We show that the L172A mutation of integrase impaired the packaging of UNG2 into viral particles. Using a primer-template DNA substrate containing G:U mispairs, we demonstrate that wild-type viral lysate has the ability to repair G:U mismatched pairs to G:C matched pairs, in contrast to UNG2-deficient viral lysate. Moreover, no correction of G:T mispairs by wild-type HIV-1 viral lysate was observed, which argues for the specificity of the repair process. We also show that UNG2 physically associates with the viral reverse transcriptase enzyme. Altogether our data indicate for the first time that a uracil repair pathway is specifically associated with HIV-1 viral particles. However, the molecular mechanism of this process remains to be characterized further.
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PMID:Functional role of HIV-1 virion-associated uracil DNA glycosylase 2 in the correction of G:U mispairs to G:C pairs. 1245 23

Nitric oxide (NO(*)) reacts with guanine in DNA and RNA to produce xanthine (X) as a major product. Despite its potential importance in NO(*)-mediated mutagenesis, the biochemical properties of X in polynucleotides have been relatively unexplored. We describe the synthesis and chemical characterization of xanthine-containing oligonucleotides and report on the susceptibility of X to depurination, its miscoding potential during replication by polymerases, and its recognition and excision by several members of the base excision repair (BER) family of DNA glycosylases. At neutral pH, X was found to be only slightly less stable than guanine to depurination (k(X)/k(G) = 1.19), whereas at pH <or= 4 the depurination rate exceeded that of G by more than an order of magnitude. HIV-1 RT inserted dCTP and dTTP with approximately equal frequencies opposite X in a DNA template, whereas DNA Pol 1(KF(-)) preferentially inserted dCTP. Several DNA glycosylases were found to excise X specifically in X.C base pairs, whereas activity toward X.G, X.A, or X.T pairs was detected only for AlkA. The order of reactivity of glycosylases for the removal of X.C base pairs was found to be AlkA > Mpg > Nth > Fpg. Implications of these results for the induction of mutations by nitric oxide are discussed.
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PMID:Stability, miscoding potential, and repair of 2'-deoxyxanthosine in DNA: implications for nitric oxide-induced mutagenesis. 1265 65

This study was aimed to apply an LC-MS-MS method previously developed for intracellular nucleoside reverse transcriptase inhibitors-triphosphate (NRTI-TPs) to the determination of natural deoxyribonucleotides (dNTPs) in human peripheral blood mononuclear cells. The LC-MS-MS method was directly used in assay of dATP and dTTP. Interferences by ribonucleotides (rNTPs) prevented direct application to the two other analytes: dGTP and dCTP. A periodate oxidation procedure was therefore optimized to remove rNTPs from the cell medium in order to quantitate dCTP and dGTP. The determination of the intracellular ratio of NRTI-TP/dNTP in HIV-infected patients now involves use of the same chromatographic system for simultaneous assay of several analytes.
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PMID:Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals. 1274 19

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k(pol): 22-fold (from 7.2 to 0.33 s(-1)) for K65R RT and 84-fold (from 7.2 to 0.086 s(-1)) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.
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PMID:A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions. 1504 78

Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7- (T7-) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts. dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased Km and attenuated kcat. Fluorescence estimates of Kd and pre-steady-state kinetic measurements of koff showed no significantly decreased affinity of T7- with the adducted oligonucleotides or the dNTP. Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme.DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always.
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PMID:Kinetics of nucleotide incorporation opposite DNA bulky guanine N2 adducts by processive bacteriophage T7 DNA polymerase (exonuclease-) and HIV-1 reverse transcriptase. 1553 46

A series of six oligonucleotides with dihydrodiol epoxide metabolites of the polycyclic aromatic hydrocarbons (PAHs) benz[a]anthracene and benzo[a]pyrene attached to adenine N6 and guanine N2 atoms were prepared and studied with the processive bacteriophage DNA polymerase T7, exonuclease- (T7-). HIV-1 reverse transcriptase was much less efficient in polymerization than T7-. Benz[a]anthracene and benzo[a]pyrene adducts strongly blocked incorporation of dTTP and dCTP opposite the A and G derivatives, respectively. dATP was preferentially incorporated in all cases. Steady state kinetic analysis indicated that the low catalytic efficiency with adducted DNA was due to both increased K(m) and lowered k(cat) values. Some differences due to PAH stereochemistry were observed. Fluorescence estimates of K(d) and presteady state kinetic measurements of k(off) showed no major decrease in the affinity of T7- with damaged DNA substrates or with dNTPs. Presteady state kinetics showed a lack of the normal burst kinetics for dNTP incorporation with all PAH-DNA derivatives. These results indicate that the rate-limiting step is at or before the step of phosphodiester bond formation; release of the oligonucleotide is no longer the slowest step. Thio elemental effects (substitution of alpha-oxygen with sulfur) were relatively small, in contrast to previous work with T7- and 8-oxo-7,8-dihydroguanine. The effect of these bulky PAH adducts is either to attenuate rates of conformational changes or to introduce an additional conformation problem but not to alter the inherent affinity of the polymerase for DNA or dNTPs.
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PMID:Kinetics of nucleotide incorporation opposite polycyclic aromatic hydrocarbon-DNA adducts by processive bacteriophage T7 DNA polymerase. 1572 Jan 47

To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dye-specific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section.
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PMID:Gene expression profiling of HIV-1 infection using cDNA microarrays. 1606 97

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.
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PMID:Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. 1727 99

Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.
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PMID:Mechanisms by which the G333D mutation in human immunodeficiency virus type 1 Reverse transcriptase facilitates dual resistance to zidovudine and lamivudine. 1796 7


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