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)

In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.
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PMID:Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity. 754 45

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

Furin is a subtilisin-like eukaryotic serine endoprotease which processes proproteins to biologically active proteins and peptides. Also, the envelope proteins of viruses, such as influenza and HIV viruses, need to be processed by furin for infectivity. This enzyme has a consensus substrate specificity for Arg-Xxx-Lys/Arg-Arg at the cleavage site. Two kinds of transition state analog peptides were designed and tested in vitro with furin. The ketomethylene series, psi (COCH2), have Ki's in the submicromolar range; the aminomethyl aminomethyl ketone series, psi(COCH2NH), have Ki's in the nanomolar range. The best inhibitor is Dec-Arg-Val-Lys-Arg-CH2-Ala-Val-Gly-NH2 (2c) with a Ki of 3.4 nM.
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PMID:Synthesis of tight binding inhibitors and their action on the proprotein-processing enzyme furin. 756 36

The third variable domain (V3 domain) of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 contains a substantial number of positively charged amino acid residues. We previously demonstrated that mutation of basic amino acid residues at position 303, 306, 309, 313, and 325 in the V3 domain of HIV-1 strain NL4-3 resulted in a dramatic elimination of both virus infectivity and syncytium-inducing ability. Mutations of arginine at position 302 to serine (R302S) or lysine at position 320 to glutamine (K320Q) had variable effects on infectivity for a panel of T cell lines tested. These mutations are located on opposite sides of the Gly-Pro-Gly-Arg-Ala sequence in the center of the V3 domain. The R302S and K320Q mutations allowed us to determine if these basic residues are important for virus neutralization by polyanionic compounds. Dextran sulfate and heparin inhibited the cytopathogenicities of both mutants for MT-4 cells, although their 50% antiviral effective doses were slightly higher than those required to achieve complete protection against wild-type HIV-1NL4-3 replication. This result emphasizes that the basic amino acids of Arg302 and Lys320 are not essential for the inhibitory effect of dextran sulfate and heparin on HIV-1 infection.
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PMID:Single basic amino acid substitutions at position 302 or 320 in the V3 domain of HIV type 1 are not sufficient to alter the antiviral activity of dextran sulfate and heparin. 757 13

Functional and structural studies were made to assess whether a class of antiviral agents targets the N-terminal domain of the glycoprotein 41,000 (gp41) of human immunodeficiency virus type 1 (HIV-1). Previous experiments have shown that the amino-terminal peptide (FP-I; 23 amino acids, residues 519-541) of HIV-1 gp41 is cytolytic to both human erythrocytes (non-CD4+ cells) and Hut-78 cells (CD4+ lymphocytes). Accordingly, FP-I-induced hemolysis may be used as a surrogate assay for evaluating the role of the N-terminal gp41 domain in HIV-cell interactions. Here, we studied the blocking of FP-I-induced lysis of erythrocytes by the following anti-HIV agents: (1) IgG [i.e., anti-(518-541) IgG] raised to an immunoconjugate of Arg-FP-I, (2) apolipoprotein A-1 (apo A-1) and a peptide based on apo A-1, (3) dextran sulfate, (4) gp41 peptide (residues 637-666), and (5) anionic human serum albumins. Dose-response curves indicated that their relative potency in inhibiting FP-I-induced hemolysis was approximately correlated with their previously reported anti-HIV activity. Electron spin resonance (ESR) studies showed that FP-I spin labeled at the N-terminal alanine binds to anti-(518-541) IgG, dextran sulfate, and anionic albumins. The high in vitro antiviral activity and low cytotoxicity of these agents suggest that blocking membrane-FP-I interactions offers a novel approach for AIDS therapy or prophylaxis.
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PMID:Antivirals that target the amino-terminal domain of HIV type 1 glycoprotein 41. 757 27

NMR spectroscopy has been used to solve the three-dimensional solution structure of a minimal RNA-binding domain of the Rev protein from the human immunodeficiency virus (type 1), an essential regulatory protein for viral replication. The presence of 10 arginine residues in the 17-residue peptide Rev34-50 caused significant problems in assignment of the NMR spectra. To improve spectral resolution, the peptide was synthesized with an alanine replacing a nonessential arginine and with selectively 15N-labeled residues. Contrary to Chou-Fasman modeling predictions an alpha-helix was detected in both water and 20% trifluoroethanol (TFE) and was found to span residues that constitute the RNA-binding and nuclear-localizing domains of Rev. The sequence-specific information provided by the NMR data gives a full description of the solution conformation of Rev34-50 which serves as a template for investigating binding of the peptide to RNA from the Rev response element (RRE). Preliminary modeling suggests that the helix can fit neatly into the expanded major groove of the RRE where interactions between the peptide side chains and the RNA can be identified. These data may aid the construction of a suitable pharmacophore model for the rational design of molecules that block Rev-RNA binding and inhibit HIV replication.
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PMID:NMR solution structure of the RNA-binding peptide from human immunodeficiency virus (type 1) Rev. 759 17

The matrix protein (MA) of human and simian immunodeficiency viruses (HIV and SIV) is encoded by the amino-terminal region of the Gag precursor and has been suggested to be involved in different processes during the early and late stages of the virus life cycle. The MA protein of SIV contains three cysteine residues at positions 57, 83, and 87, which are also highly conserved among HIV-2 isolates. In order to study the functional significance of these residues in virus morphogenesis, a series of mutations affecting the cysteines of SIV MA were introduced into a gag-protease construct and expressed in the vaccinia vector system. The MA mutants were assayed for their ability to synthesize and process the Gag polyprotein precursor as well as to release particles into the culture medium. In addition, the incorporation of the envelope glycoprotein (Env) into the Gag-made particles was investigated. Substitution of alanine for cysteine 87 had little effect on particle release and Env glycoprotein association. By contrast, the individual replacement of cysteines 57 or 83 by alanine, as well as the simultaneous mutation of cysteines 83 and 87, significantly reduced the ability of Gag polypeptides to produce extracellular particles. Assembly into particles appeared to be also affected, albeit to a lesser extent, when both cysteines 57 and 83 were replaced by alanine. Furthermore, substitution of cysteine 83 in the SIV MA domain was found to be detrimental to Gag polyprotein processing. Analysis of the Env glycoprotein association with recombinant particles revealed that this process was moderately affected in the case of the double mutants lacking cysteines 57 and 83, or cysteines 57 and 87, and the cysteine-minus triple mutant. Our results suggest that the conserved cysteines 57 and 83 in the MA domain are important for efficient SIV Gag particle production.
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PMID:Mutational analysis of the conserved cysteine residues in the simian immunodeficiency virus matrix protein. 761 87

Erythropoietin (EPO) is the primary hormone responsible for the growth and maturation of red blood cells in mammals. In contrast to many other growth factors, the specificity of EPO for mature erythroid cells has lead to its development as a safe and efficacious therapeutic, EPREX. The medical benefits of EPREX have been well established in the treatment of anaemic chronic renal failure patients, anaemic HIV patients treated with AZT, cancer chemotherapy patients, and patients wishing to donate their own blood prior to elective surgery (autologous predonation). Due to the chronic nature of EPO therapy, it would be desirable to have an orally administered 'second generation' molecule. An understanding of the structural basis of the interaction of EPO with its receptor will aid in the design of an oral anaemia drug. In this study, a series of mutations have been generated in a truncated form of the receptor comprising the extracellular region, termed EPO binding protein (EBP). One mutant, in which alanine replaces phenylalanine at position 93 (F93A) has a 500-fold reduction in binding compared to wild-type EBP. A neutralizing anti-EBP antibody binds poorly to the F93A mutant, while a non-neutralizing anti-EBP antibody binds wild-type and F93A equally well. Information from this mutational analysis can be applied to a receptor 3-D model and ultimately used in drug development.
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PMID:Erythropoietin receptor: application in drug development. 764 2

The intrinsic fluorescence of tyrosine increases by a factor of approximately two when the carboxy group is liberated from a peptide bond by hydrolysis. The increase in fluorescence provides a novel way to monitor the hydrolysis of native tyrosine peptides that contain only proteinogenic amino acids. Thus, for example, the hydrolysis by HIV-1 proteinase of a heptapeptide viral protein fragment gag129-135, Ser-Gln-Asn-Tyr-Pro-Ile-Val, was followed continuously at excitation and emission wavelengths 275 and 305 nm. The fluorescence increase is magnified by at least a factor of a thousand when a resonance energy quencher, such as paranitrophenylalanine, is in the vicinity. For example, the peptide Lys-Ala-Arg-Val-Tyr-Phe(p-NO2)-Glu-Ala-Nle-NH2 [Richards et al. (1990) J. Biol. Chem. 265, 7733], widely used for spectrophotometric assays of the HIV-1 proteinase, yields a substrate:product fluorescence ratio greater than 1:1000. Tyrosine-containing substrates of pepsin and trypsin showed similar behavior. The detection limit of the present method is at least one order of magnitude lower than absorbance assays of p-nitrophenylalanine peptides.
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PMID:Increase in fluorescence upon the hydrolysis of tyrosine peptides: application to proteinase assays. 766 86

Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated by the HIV-1-specific inhibitors bis-heteroarylpiperazine (BHAP), 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-on e (TIBO) R82913, nevirapine, and the N3-methylthymine derivative of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO-m3T), as single agents or in combination, at escalating concentrations. When used individually, the compounds led to the emergence of drug-resistant virus strains within two to five subcultivations. The resulting strains were designated HIV-1/BHAP, HIV-1/TIBO, HIV-1/Nev, and HIV-1/TSAO-m3T, respectively. The mutant viruses showed the following amino acid substitutions in their reverse transcriptase (RT): Leu-100-->Ile for HIV-1/BHAP; Lys-103-->Asn for HIV-1/TIBO; Val-106-->Ala for HIV-1/Nev; and Glu-138-->Lys for HIV-1/TSAO-m3T. Both the Tyr-181-->Cys and Val-106-->Ala mutations were found in another mutant emerging following treatment with nevirapine at escalating concentrations. The BHAP-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and TSAO-m3T, whereas the TSAO-m3T-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and BHAP. When different pairs of nonnucleoside RT inhibitors (i.e., BHAP plus TSAO-m3T, nevirapine plus TSAO-m3T, TIBO plus TSAO-m3T, nevirapine plus TIBO, and BHAP plus nevirapine) were used, resistant virus emerged as fast as with single-drug therapy. In all cases the Tyr-181-->Cys mutation appeared; the virus showed markedly reduced sensitivity to all HIV-1-specific inhibitors but retained sensitivity to 2',3'-dideoxynucleoside analogs such as zidovudine, ddC, and ddI. Our findings argue against simultaneous combination of two different nonnucleoside RT inhibitors that are unable to inhibit HIV-1 mutant strains containing the Tyr-181-->Cys mutation when administered as single drugs.
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PMID:Treatment of human immunodeficiency virus type 1 (HIV-1)-infected cells with combinations of HIV-1-specific inhibitors results in a different resistance pattern than does treatment with single-drug therapy. 768 22


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