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)

A convenient and straightforward synthesis of an amino acid analog, [p-(N-alpha-Fmoc-L-aspartic acid-beta-amido)benzyl]-EDTA tetra-tert-butyl ester, compatible with Fmoc solid phase peptide synthesis strategy is described. This reagent was used to incorporate p-aminobenzyl-EDTA at an internal sequence position in an HIV-1 Tat protein fragment. After cleavage from the resin and standard deprotection, the peptide was purified by high-performance liquid chromatography and characterized by mass spectrometry. Through this methodology, flexible linkers of different lengths and containing various structures can be placed between the alpha-carbon backbone of peptides and metal chelates. These peptides will provide a new class of affinity cleaving reagents that can be directed against protein and nucleic acid targets.
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PMID:Synthesis of an amino acid analogue to incorporate p-aminobenzyl-EDTA in peptides. 909 68

Viral populations in a human immunodeficiency virus type 1 (HIV-1)-infected individual behave as a quasispecies with a rated distribution of fitness variants. Fitness distributions in naturally occurring viral populations have been difficult to study due to the lack of markers for individual virus clones and complicating inter- and intrahost factors like the presence of multiple cell types with distinct tropisms, differences in route of transmission, and intervening immunity. Here, we quantitated the relative fitness in vivo of three subpopulations of HIV-1 marked by mutations at codons 41 and 215 of reverse transcriptase (RT) directly related to zidovudine resistance in an untreated individual who was infected by a zidovudine-resistant strain transmitted from a donor on therapy. The transmission event did not have a substantial impact on the distribution of mutants within the dominant virus population replicating to high levels in the recipient. The evolution of the RT gene was monitored for 20 months. All 102 clones obtained from the donor and the recipient at the different time points contained the M41L mutation, which is associated with a fourfold reduction in zidovudine sensitivity. The leucine at position 41 was stable, although it was encoded by TTG and CTG triplets that fluctuated in abundance partially due to founder effects of clones with nonsilent mutations at codon 215. Of the three subpopulations in the patient, distinguished by a tyrosine (TAC), aspartic acid (GAC), or serine (TCC) at the 215 position of RT, the relative fitness of the GAC variant was calculated to be 10 to 25% higher than the initial TAC variant, and the relative fitness of the TCC variant was 1% higher than that of the GAC variant. Similar to other RNA viruses, lentivirus populations like HIV-1 in patients with a high virus load apparently consist of a broader spectrum of fitness variants than the 1 to 2% fitness difference sufficient for significant replicative advantage.
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PMID:Broad spectrum of in vivo fitness of human immunodeficiency virus type 1 subpopulations differing at reverse transcriptase codons 41 and 215. 915 39

The CC-chemokine receptor CCR5 is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of CD4+ cells and interacts directly with the viral surface glycoprotein gp120. Although receptor chimera studies have provided useful information, the domains of CCR5 that function for HIV-1 entry, including the site of gp120 interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of CCR5 to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish CCR5-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair Env-mediated membrane fusion and the gp120-CCR5 interaction. Of these three residues, only D11 is necessary for CC-chemokine-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites on CCR5 are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal CCR5 domain.
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PMID:Amino-terminal substitutions in the CCR5 coreceptor impair gp120 binding and human immunodeficiency virus type 1 entry. 942 Feb 25

The cellular tropism of human immunodeficiency virus type 1 (HIV-1) is dependent on utilization of specific chemokine co-receptor: macrophage-tropic/non-syncytium-inducing (NSI) viruses use CCR5, whereas T-cell tropic/syncytium-inducing (SI) viruses preferentially use CXCR4. We have analyzed co-receptor usage of 24 phylogenetically distinct primary HIV-1 isolates representing group M (clades A-F) and group O with known SI and NSI phenotype, using lymphocytes from donor with nonfunctional CCR5 (CCR5-/-; homozygous 32-bp deletion). While all SI isolates infected CCR5-/- lymphocytes (and hence do not require CCR5 for viral entry), all NSI isolates, regardless of clade, did not infect CCR5-/- lymphocytes. Thus, CCR5 expression is required for infection with NSI isolates and the CCR5 usage is independent of viral genotype. To localize the viral determinant involved in CCR5 binding, the V3 sequences across the clades were aligned based on the CCR5 usage. There were conserved uncharged residues at position 11 of V3 (mostly serine/glycine) and negatively charged residues at residue 25 (mostly glutamic/aspartic acid) among all isolates that used CCR5, whereas substitution with arginine or glutamine at these two positions led to usage of a co-receptor other than CCR5. This analysis led us to identify a consensus motif S/GXXXGPGXXXXXXXE/D within the V3 loop that predicts CCR5 co-receptor usage. Most isolates, with exception of one isolate, containing the conserved motif and predicted to utilize CCR5 indeed had an absolute requirement of CCR5 expression for infectibility. Site-directed mutagenesis in the infectious molecular clone further confirmed these results. Taken together, these data provide evidence that sequences within the V3 loop provide important residues that might be directly or indirectly involved in binding to a CCR5 co-receptor.
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PMID:CCR5 coreceptor usage of non-syncytium-inducing primary HIV-1 is independent of phylogenetically distinct global HIV-1 isolates: delineation of consensus motif in the V3 domain that predicts CCR-5 usage. 944 92

The HIV-1 transframe region (TFR) is between the structural and functional domains of the Gag-Pol polyprotein, flanked by the nucleocapsid and the protease domains at its N and C termini, respectively. Transframe octapeptide (TFP) Phe-Leu-Arg-Glu-Asp-Leu-Ala-Phe, the N terminus of TFR, and its analogues are competitive inhibitors of the action of the mature HIV-1 protease. The smallest, most potent analogues are tripeptides: Glu-Asp-Leu and Glu-Asp-Phe with Ki values of approximately 50 and approximately 20 microM, respectively. Substitution of the acidic amino acids in the TFP by neutral amino acids and d or retro-d configurations of Glu-Asp-Leu results in an >40-fold increase in Ki. Protease inhibition by Glu-Asp-Leu is dependent on a protonated form of a group with a pKa of 3.8; unlike other inhibitors of HIV-1 protease which are highly hydrophobic, Glu-Asp-Leu is extremely soluble in water, and its binding affinity decreases with increasing NaCl concentration. However, Glu-Asp-Leu is a poor inhibitor (Ki approximately 7.5 mM) of the mammalian aspartic acid protease pepsin. X-ray crystallographic studies at pH 4.2 show that the interactions of Glu at P2 and Leu at P1 of Glu-Asp-Leu with residues of the active site of HIV-1 protease are similar to those of other product-enzyme complexes. It was not feasible to understand the interaction of intact TFP with HIV-1 protease under conditions of crystal growth due to its hydrolysis giving rise to two products. The sequence-specific, selective inhibition of the HIV-1 protease by the viral TFP suggests a role for TFP in regulating protease function during HIV-1 replication.
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PMID:Hydrophilic peptides derived from the transframe region of Gag-Pol inhibit the HIV-1 protease. 948 57

Sequences of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) domain were determined by direct sequencing of HIV-1 RNA in successive plasma samples from eight seroconverting patients infected with virus bearing the T215Y/F amino acid substitution associated with zidovudine (ZDV) resistance. At baseline, additional mutations associated with ZDV resistance were detected. Three patients had the M41L amino acid change, which persisted. Two patients had both the D67N and the K70R amino acid substitutions; reversion to the wild type was seen at both positions in one of these patients and at codon 70 in the other one. Reversion to the wild type at codon 215 was observed in only one of eight patients. Unusual amino acids, such as aspartic acid (D) and cysteine (C), appeared at position 215 in four patients during follow-up. These variants isolated by coculturing were sensitive to ZDV. Overgrowth of these variants suggests that they have better fitness than the original T215Y variant. Intraindividual nucleoside substitutions over time were 10 times more frequent in codons associated with ZDV resistance (41, 67, 70, 215, and 219) than in other codons of the RT domain. The predominance of nonsynonymous substitutions observed over time suggests that most changes reflect adaptation of the RT function. The variance in sequence evolution observed among patients, in particular at codon 215, supports a role for chance in the evolution of the RT domain.
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PMID:Switch to unusual amino acids at codon 215 of the human immunodeficiency virus type 1 reverse transcriptase gene in seroconvertors infected with zidovudine-resistant variants. 955 30

The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3'-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 10(3)- to 10(4)-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721-728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per microg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3'-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.
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PMID:Mutations in the human immunodeficiency virus type 1 integrase D,D(35)E motif do not eliminate provirus formation. 957 31

The metabolism of different phosphoramidate prodrugs of d4T-MP, in which the phosphate group is linked to a phenyl group and the alkyl ester of an amino acid was studied in crude CEM cell extracts. Significant (80-100%) conversion to the amino acyl d4T-MP metabolite was obtained with derivatives containing L-alanine or methyl-L-aspartic acid. A lower degree of conversion was seen with derivatives containing L-phenylalanine, L-methionine, methyl-L-glutamic acid or L-leucine. Derivatives containing D-alanine, beta-alanine, glycine, L-valine or L-lactate showed no conversion to the amino acyl d4T-MP metabolite. Overall, there was a close correlation between the anti-HIV activity of these prodrugs and their conversion rate to the amino acyl d4T-MP metabolite. Our data suggest that the enzymes involved in the formation of the amino acyl d4T-MP metabolite have a rather stringent specificity for L-alanine as the amino acid moiety. In addition, these enzymes were found to be markedly species-dependent, their activities being highest in mouse serum, followed by guinea pig serum, but only minimal in human serum. Mouse serum therefore appears to be the medium of choice to isolate and identify the enzymes that are involved in the metabolism of these phosphoramidate prodrugs.
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PMID:Metabolism and anti-HIV activity of phosphoramidate derivatives of D4T-MP with variations in the amino acid moiety. 959 64

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.
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PMID:Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein. 962 70

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.
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PMID:Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100. 965 78


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