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)

The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
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PMID:Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity. 769 May 4

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp41 as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.
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PMID:A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. 769 82

Of the class of the 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine (HEPT) derivatives, several congeners were found to inhibit (at 50% effective concentrations ranging from 0.02 to 0.6 microgram/ml) the replication of mutant human immunodeficiency virus type 1 (HIV-1) strains that had been selected for resistance against bis(heteroaryl)piperazine, tetrahydroimidazo[4,5,1-jk] [1,4]benzodiazepin-2(1H)-thiones (TIBO), nevirapine, [2',5'-bis-O-(tert- butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino- 1'',2'' -oxathiole-2'',2''-dioxide) (TSAO), or pyridinone and showed amino acid substitutions at positions 100, 103, 106, 138, and 181, respectively. When HIV-1 strains were selected for resistance against three different HEPT derivatives [i.e., HEPT and its derivatives 5-ethyl-1-ethoxymethyl-6-benzyluracil(E-EBU) and 5-ethyl-1-ethoxymethyl-6-(3,5-dimethylbenzyl)uracil (EBU-dM)], HEPT selected for the mutation 188-Tyr-->His, E-EBU for 181-Tyr-->Cys, and E-EBU-dM for 106-Val-->Ala, in the reverse transcriptase of the mutant viruses. These virus strains showed markedly decreased sensitivity to HEPT derivatives. Moreover, the HEPT-resistant virus strains also proved cross-resistant to virtually all other HIV-1-specific inhibitors, including TIBO, nevirapine, and TSAO.
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PMID:Human immunodeficiency virus type 1 drug-resistance patterns with different 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives. 769 68

The transmembrane protein of human immunodeficiency virus type 1 (HIV-1) contains a leucine zipper-like (hydrophobic heptad) repeat which has been predicted to form an amphipathic alpha helix. To evaluate the potential of the hydrophobic heptad repeat to induce protein oligomerization, this region of gp41 has been cloned into the bacterial expression vector pRIT2T. The resulting plasmid, pRIT3, expresses a fusion protein consisting of the Fc binding domain of monomeric protein A, a bacterial protein, and amino acids 538 to 593 of HIV-1 gp41. Gel filtration chromatography demonstrated the presence of oligomeric forms of the fusion protein, and analytical centrifugation studies confirmed that the chimeric protein formed a higher-order multimer that was greater than a dimer. Thus, we have identified a region of HIV-1 gp41 which is capable of directing the oligomerization of a fusion protein containing monomeric protein A. Point mutations, previously shown to inhibit the biological activity of the HIV-1 envelope glycoprotein, have been engineered into the segment of gp41 contained in the fusion protein, and expressed mutant proteins were purified and analyzed via fast protein liquid chromatography. A point mutation in the heptad repeat, which changed the central isoleucine to an alanine, caused a significant (> 60%) decrease in oligomerization, whereas changing the central isoleucine to aspartate or proline resulted in almost a complete loss of oligomerization. Deletions of one, two, or three amino acids following the first isoleucine also resulted in a profound decrease in oligomerization. The inhibitory effects of the mutations on oligomer formation correlated with the effects of the same mutations on envelope glycoprotein-mediated fusion. A possible role of the leucine zipper-like region in the fusion process and in an oligomerization event distinct from assembly of the envelope glycoprotein complex is discussed.
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PMID:Oligomerization of the hydrophobic heptad repeat of gp41. 770 97

An expression system has been established in Escherichia coli to facilitate the preparation of the HIV-1 capsid protein in amounts sufficient for structural analysis. A plasmid vector pTCA5, containing the gene for the recombinant HIV-1 capsid protein rp24 under the control of the lambda-PR-promoter, was constructed which gave an expression product that spanned 234 amino acid residues. It differs at the N-terminus from the authentic sequence in that the residues Pro-Ile- are replaced by Met-Asn-Ser-Ala-Met-. Recombinant p24 was produced, as inclusion bodies in E. coli LE392 containing pTCA5, at a level of approximately 15% of the total cellular protein. After dissolution of the inclusion bodies in the acidic urea system, the protein was easily reconstituted in a soluble state by dialysis. The yield of reconstituted and purified protein was 12 mg per liter in rich medium. Recombinant rp24 consists of about 40% alpha-helix and 10% beta-sheet from circular dichroism measurements and the two cysteine residues, within the rp24 sequence, are bridged by a disulfide bond.
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PMID:A recombinant human immunodeficiency virus type-1 capsid protein (rp24): its expression, purification and physico-chemical characterization. 771 31

vpr is one of the auxiliary genes of human immunodeficiency virus type 1 (HIV-1) and is conserved in the related HIV-2/simian immunodeficiency virus lentiviruses. The unique feature of Vpr is that it is the only nonstructural protein incorporated into the virus particle. Secondary structural analysis predicted an amphipathic alpha-helical domain in the amino terminus of Vpr (residues 17-34) which contains five acidic and four leucine residues. To evaluate the role of specific residues of the helical domain for virion incorporation, mutagenesis of this domain was carried out. Substitution of proline for any of the individual acidic residues (Asp-17 and Glu-21, -24, -25, and -29) eliminated the virion incorporation of Vpr and also altered the stability of Vpr in cells. Conservative replacement of glutamic residues of the helical domain with aspartic residues resulted in Vpr characteristic of wild type both in stability and virion incorporation, as did substitution of glutamine for the acidic residues. In contrast, replacement of leucine residues of the helical domain (residues 20, 22, 23, and 26) by alanine eliminated virion incorporation function of Vpr. These data indicate that acidic and hydrophobic residues and the helical structure in this region are critical for the stability of Vpr and its efficient incorporation into virus-like particles.
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PMID:Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation. 773 85

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

Upon in vitro processing of the recombinant HIV-1/gag p24 protein, expressed in Escherichia coli as a fusion protein, by HIV-1 protease, a cleavage site within the staphylococcal protein A fusion partner was found. N-terminal sequencing of the protein A fragments showed that HIV-1 protease cleavage occurred between phenylalanine-235 and tyrosine-236 within the sequence Gln-Asn-Ala-Phe/Tyr-Glu-Ile-Leu (QNAF/YEIL) in the IgG-binding domain C of the protein A encoded by the pRIT2T fusion gene vector (Pharmacia). Results presented here have proven that the protease-sensitive site is viable in vitro on the protein A alone and other chimeric protein, protein A/beta-galactosidase. A possible significance of this phenomenon in biotechnology work is discussed.
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PMID:Staphylococcal protein A is a novel heterologous substrate for the HIV-1 protease. 776 14

A major problem in the development of antiviral therapies for AIDS has been the emergence of drug resistance. We report an analysis of the structure of a Val 82 to Ala mutant of HIV-1 proteinase complexed to A-77003, a C2 symmetry-based inhibitor. Modelling studies predicted that the V82A mutation would result in decreased van der Waals' interactions with the phenyl rings of A-77003 in both S1 and S1' subsites. Unexpected rearrangements of the protein backbone, however, resulted in favourable re-packing of inhibitor and enzyme atoms in the S1 but not the S1' subsite. This analysis reveals the importance of enzyme flexibility in accommodating alternate packing arrangements, and can be applied to the re-design of inhibitors targeted to drug resistant variants which emerge in the clinic.
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PMID:Structural basis of drug resistance for the V82A mutant of HIV-1 proteinase. 777 92

Mutations of human immunodeficiency virus type 1 (HIV-1) protease at four positions, Val82, Asp30, Gly48, and Lys45 were analyzed for the resulting effects on kinetics and inhibition. In these mutants, Val82 was substituted separately by Asn, Glu, Ala, Ser, Asp, and Gln; Asp30 was individually substituted by Phe or Trp; Gly48 by His, Asp, and Tyr, respectively; and Lys45 by Glu. By examination of the inhibition of a single inhibitor, the differences in Ki values between the native and mutant enzymes can range from very large to insignificant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of Ki changes was observed for a group of inhibitors: Thus, how much the inhibition changes from the wild-type enzyme to a mutant is dependent on both the mutation and the inhibitor. The examination of Ki changes of inhibitors with closely related structures binding to Val82 mutants also reveals that the change of inhibition involves subsites in which Val82 is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of the mutants, the kcat and Km values of many Val82 mutants and a Lys45 mutant are comparable to the native enzyme. Surprisingly, Gly48 mutations produce enzymes with catalytic efficiency superior to that of the wild-type enzyme by as much as 10-fold. Modeling of the structure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region of the mutants. The examination of the relative changes of inhibition and catalysis of mutants suggests that some of the Val82 and Gly48 mutants are potential resistance mutants. However, the resistance is specific with respect to individual inhibitors.
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PMID:Effect of point mutations on the kinetics and the inhibition of human immunodeficiency virus type 1 protease: relationship to drug resistance. 782 64


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