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

Nuclear Overhauser effect experiments were performed at 500 MHz to determine the conformations of AZTTP and dTTP when bound to HIV-1 reverse transcriptase. The conformations of both ligands were found to be similar in the bound state. The orientation of the glycosidic angle is anti (chi = -120 degrees +/- 12 for AZTTP and -110 degrees +/- 12 for dTTP), gamma is +sc and the pucker of the 3'-azido-2',3'-dideoxy- and 2'-deoxyribose rings is predominantly C4' exo (P = 60 degrees +/- 10 for AZTTP and 55 degrees +/- 8 for dTTP). These results indicate that the unusual C4'endo/C3'exo pucker (P = 215 degrees) reported for the dideoxyribose ring of AZT in the solid state does not play a role in the interaction of HIV-1 reverse transcriptase with AZTTP.
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PMID:A comparison of the conformations of the 5'-triphosphates of zidovudine (AZT) and thymidine bound to HIV-1 reverse transcriptase. 768 61

Six affinity reagents containing chemically reactive groups, either on the phosphate residue at the 5'-end or on the 5'- or 3'-end internucleoside phosphate linkages of the oligothymidylate primers, were used to covalently modify the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT). After covalent binding of these modified primer analogs to the enzyme, the addition of [alpha-32P]dTTP, in the presence of a complementary template, led to elongation of the primer. This reaction was catalyzed by the active site of the enzyme carrying the covalently bound primer. The relative efficiency of labeling of the p66/p51 heterodimer compared to the p66/p66 and p51/p51 homodimers of HIV-1 RT was in agreement with the previously determined affinity of the various enzyme forms toward different primers. The analogues preferentially modified the p66 subunit of the HIV-1 RT heterodimer. The labeling of all RT forms by synthetic primer analogues showed significant and specific competition by the natural primer of HIV-1 RT, tRNA(Lys). In addition, the kinetics of inactivation of RT by primer analogues was studied. The affinity of the enzyme to those derivatives in the presence of poly(A) template was about 5-10 times higher than in the absence of template. Moreover, the maximal rates of HIV-1 RT inactivation by analogues in the absence of template were 3-4 times higher. Our results suggest that the mechanism of oligonucleotide primer binding to HIV-1 RT is different in the presence or absence of template.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Affinity labeling and functional analysis of the primer binding domain of HIV-1 reverse transcriptase. 768 10

A minimal kinetic mechanism for HIV reverse transcriptase (RT)-catalyzed RNA-dependent and DNA-dependent DNA polymerization was determined by pre-steady-state kinetic methods to be: [formula: see text] where E, TP, dNTP, and PPi are RT, template-primer, 2'-deoxynucleoside 5'-triphosphate, and inorganic pyrophosphate, respectively. Defined sequence template-primers that encode for incorporation of dTTP were prepared by annealing either a 44-mer RNA template or a 44-mer DNA template (of the same sequence) to a 21-mer DNA primer (r44:d21-mer and d44:d21-mer, respectively). The values of the above kinetic constants were determined for dTMP and 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP) incorporation into both template primers. The kcat and Km values calculated from these kinetic constants were similar to the values directly determined from steady-state experiments. Further, the net rate constants for processive incorporation of three successive nucleotides into the r44:d21-mer were similar indicating that a rate-determining step did not follow catalysis. A 20-fold difference in the rate constants (kp) for incorporation of dTMP into the r44:d21-mer versus the d44:d21-mer was largely responsible for the difference in the calculated processivity numbers of 340 and 5, respectively. Finally, the rate constant for pyrophosphorolysis of the 3'-AZTMP-terminated r44:d21-mer (kpyro) was similar to the rate constant for dissociation of the chain-terminated template primer from the enzyme (koff) indicating that millimolar concentrations of intracellular inorganic pyrophosphate would be required for pyrophosphorolysis of AZTMP-terminated retroviral genomes.
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PMID:Human immunodeficiency virus reverse transcriptase. A kinetic analysis of RNA-dependent and DNA-dependent DNA polymerization. 768 54

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.
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PMID:Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label. 768 65

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.
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PMID:Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. 769 3

The sequence of 2350 nucleotides in the env and IN regions of a group O HIV-1 genome which is hypermutated throughout its entirety was compared to the equivalent sequence of a nonhypermutated genome from the same isolate. Almost 30% of G residues were affected by G-->A transitions. As previously reported, transitions occurred mainly at GpA and GpG dinucleotides, with a marked preference for changes of the 5'-proximal G residues in poly(G) stretches. Inspection of the sequences around the hypermutation sites revealed no bias when the mutation was at the 5' G residue of a GpG dinucleotide. In contrast, a preferred context for hypermutation at the 3' G (or at single G residues) could be defined. In addition to a preference for A residues immediately downstream of hypermutated 3' G residues, C residues were underrepresented in these positions. The observed context fits well with a model whereby G-->A mutation occurs by a combination of dislocation mutagenesis at GpA dinucleotides and direct misincorporation of dTTP at the 5' G of GpG dinucleotides. Furthermore, both runs of six G residues present in the polypurine tracts (PPTs) had escaped hypermutation, despite the fact that 95% of runs of three G residues contained at least one G-->A transition. This finding suggests that genomes with hypermutated PPT motifs had been selected against and provides direct evidence that hypermutation occurs during negative-strand DNA synthesis.
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PMID:A highly defective HIV-1 group O provirus: evidence for the role of local sequence determinants in G-->A hypermutation during negative-strand viral DNA synthesis. 774 32

The effects of hydroxyurea (HU), an inhibitor of ribonucleotide reductase, on the replication of human immunodeficiency virus type 1 (HIV-1) in activated peripheral blood mononuclear cells were studied. The inhibition of HIV-1 replication by HU alone was dose dependent, with a 90% inhibitory concentration of 0.4 mM, a plasma concentration tolerated by patients with oncological diseases. HU at lower concentrations (< 0.1 mM) was found to potentiate the antiviral activity of 2',3'-dideoxyinosine (ddl), 3'-azido-2',3'- dideoxythymidine, and 2',3'-dideoxycytidine against HIV-1, with the potentiation being ddl greater than 3'-azido-2',3'- dideoxythymidine = 2',3'-dideoxycytidine. In the presence of 0.1 mM HU, the 90% inhibitory concentration of ddl was reduced by 6-fold in activated peripheral blood mononuclear cells. The potentiating effect of HU on ddl action was time dependent, with the greatest inhibition of HIV-1 growth being seen when HU was present during and after virus adsorption, i.e., apparently coinciding with the time of proviral DNA synthesis. A brief incubation of activated cells with HU and ddl at low concentrations before virus exposure reduced p24 production by > 50%. Analyses using high performance liquid chromatography and enzymatic assays suggested that the greater degree of potentiation by HU of the action of ddl, compared with the other dideoxynucleosides, is due to the more effective inhibition by HU of dATP synthesis, compared with the synthesis of the other deoxynucleoside triphosphates (dGTP, dTTP, and dCTP). The present study suggests that, for appropriate agents, pharmacological reduction of deoxynucleoside triphosphate levels represents a potential therapeutic approach for inhibition of HIV-1 replication.
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PMID:Anti-human immunodeficiency virus type 1 activity of hydroxyurea in combination with 2',3'-dideoxynucleosides. 796 58

An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.
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PMID:Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase. 822 79

The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentration which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 microM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10- to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in approximately 5-fold and approximately 15-fold lower ratios of ddCTP/dCTP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase. We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy.
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PMID:Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells. 838 46

Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT) were isolated using three methods designated A, B, and C. The three samples of wt RT were kinetically indistinguishable with respect to dTTP turnover on poly(rA).p(dT)10. However, whereas the kinetic constants for dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt protein, T215Y A exhibited a twofold increase in Km value for dTTP and a 13-fold increase in Ki value for AZTTP with respect to wt protein purified in the same manner. We further investigated this observation by studying the denaturation of wt RT by urea. The urea denaturation curves monitored by fluorescence and circular dichroism spectroscopy were not coincident with the denaturation curve monitored by enzyme activity and yielded Cm values (the concentration of urea at which 50% of the protein is denatured) of 4.1 and 2.0 M urea, respectively. The noncoincidence of the transition curves reflects two separable, sequential, noncooperative conformational changes in the molecule: (a) from a catalytically active to an inactive conformation, and (b) from a catalytically inactive to a denatured, unfolded conformation. We therefore used denaturation as detected by changes in enzyme activity to compare the conformational stability of the three samples of wt and T215Y RT A, B, and C. The Cm values for T215Y RT did not differ from those of the respective wt; however, differences in Cm values were noted depending on how the protein was isolated. This suggested that the heterogeneity of the recombinant RT was due to small differences in conformation at or near the active site.
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PMID:Recombinant human immunodeficiency virus type 1 reverse transcriptase is heterogeneous. 852 29

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