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)

Molecular models of HIV-1 protease and 21 peptide substrates with single amino acid substitutions at positions from P4 to P3' were built and compared with kinetic measurements. The crystal structure of HIV-1 protease with a peptidic inhibitor was modified to model the peptide substrate Pro-Ala-Val-Ser-Leu-Ala-Met-Thr for the starting geometry. Models were built of two reaction intermediates, HIV protease with peptide substrate and with its tetrahedral intermediate. The energy minimization used a new algorithm that increased the speed and eliminated a cut-off for non-bonded interactions. After minimization the models for substrate and tetrahedral intermediate both had root mean square deviations of 0.48 A for all atoms of the HIV protease compared to the starting crystal structure. Differences in the model structures and interaction energies for HIV protease with different substrates were analyzed. The calculated interaction energies for the 21 HIV protease-tetrahedral intermediate models gave a correlation coefficient of 0.64 with the kinetic measurements. The eight substrates with changes in the P1 and P1' residues next to the scissile bond gave the highest correlation of 0.93, while the 14 substrates with changes in P2-P2' gave a correlation coefficient of 0.86. The catalytic mechanism and factors influencing the catalytic efficiency of the different substrates are discussed in relation to the models. The predictive ability of molecular mechanics calculations is discussed in the context of the statistical mechanics analysis of the differences in free energy.
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PMID:Molecular mechanics calculations on HIV-1 protease with peptide substrates correlate with experimental data. 887 45

The nef genes of HIV-1 and SIV encode 27-34 kDa myristoylated proteins which have been shown to induce cell surface CD4 downregulation and bind to a cellular protein kinase. To identify regions of Nef important for function, structure-function correlates of HIVSF2 nef (Nef) and SIVmm239open nef (SNef) were sought by constructing Nef/SNef hybrids. Metabolic labeling with 35[S]methionine/cysteine demonstrated similar amounts of 35[S] incorporation into all but one hybrid, SNeftll, in which the C-terminus of SNef was replaced by that of Nef. The weak protein expression of SNeftll was attributable to its short half-life of approximately 45 min. Nef, SNef, and SHSNef, a hybrid containing the internal sequences of Nef and the N- and C-terminal sequences from SNef, downregulated CD4 in human CEM cells. Only Nef and SHSNef downregulated CD4 in mouse AKR1-G1 cells. Nef, SNef, and SHSNef also effectively bound phosphoproteins of MW = 62,000 and 78,000 in CEM cells. Two additional hybrids, in which the Nef sequences of SHSNef were replaced with additional SNef sequences, were essentially ineffective in both assays. Thus, in two different assays of Nef function, swapping the SIV and HIV internal nef sequences were shown to be greatly deleterious to Nef function while SHSNef remained functional.
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PMID:Structural and functional correlates between HIV-1 and SIV Nef isolates. 895 34

Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides.
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PMID:A novel Met-to-Thr mutation in the YMDD motif of reverse transcriptase from feline immunodeficiency virus confers resistance to oxathiolane nucleosides. 903 72

The retroviral proteases (PRs) have a structural feature called the flap, which consists of a short anti-parallel beta-sheet with a turn. The flap extends over the substrate binding cleft and must be flexible to allow entry and exit of the polypeptide substrates and products. We analyzed the sequence requirements of the amino acids within the flap region (positions 46-56) of the HIV-1 PR. The phenotypes of 131 substitution mutants were determined using a bacterial expression system. Four of the mutant PRs with mutations in different regions of the flap were selected for kinetic analysis. Our phenotypic analysis, considered in the context of published structures of the HIV-1 PR with a bound substrate analogs, shows that: (i) Met-46 and Phe-53 participate in hydrophobic interactions on the solvent-exposed face of the flap; (ii) Ile-47, Ile-54, and Val-56 participate in hydrophobic interactions on the inner face of the flap; (iii) Ile-50 has hydrophobic interactions at the distance of both the delta and gamma carbons; (iv) the three glycine residues in the beta-turn of the flap are virtually intolerant of substitutions. Among these mutant PRs, we have identified changes in both kcat and Km. These results establish the nature of the side chain requirements at each position in the flap and document a role for the flap in both substrate binding and catalysis.
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PMID:Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants. 912 79

We examined the phenotypic and genotypic properties of virus from the peripheral blood mononuclear cells (PBMC) and plasma of eight HIV-1-infected asymptomatic patients before and during monotherapy with the proteinase inhibitor saquinavir. Susceptibility of primary isolates to drug was assessed in PBMC culture by deriving IC50 and IC90 values. The observed increases in IC50 and IC90 after approximately one year of therapy with a dosage of 600 mg tds suggests the presence of virus resistant to saquinavir in vivo. The magnitude of this altered susceptibility ranged from three-fold to in one case 100-fold. In two patients a greater than eight-fold decrease in susceptibility to saquinavir was observed. Sequencing of the proteinase genes in viral RNA obtained from patient plasma and/or PBMC was carried out by PCR in parallel with sensitivity testing. In each case between nine and 12 clones were analysed. In the two patients from whom virus had greater than eight-fold reduction in susceptibility, a point mutation was observed in the viral proteinase (Leu90--> Met/Ile). Further mutations were observed at residues 36, 71 and 84 in these subjects. In a third patient, in whom an eight-fold increase in HIV IC50 of saquinavir was observed, no mutations were detected in the proteinase; sequencing of proteinase cleavage sites in viral gag-pol revealed no significant mutations. In no patient was a Gly48-->Val mutation observed, although this has been associated with resistance in vitro. The Leu90-->Met mutation was observed in five subjects, but a greater than eight-fold phenotypic change in antiviral susceptibility was seen in only two of these. Hence, in vivo, the Leu90-->Met but not the Gly48-->Val mutation is necessary, but not sufficient, for phenotypic resistance to saquinavir in HIV.
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PMID:Emergence of resistant variants of HIV in vivo during monotherapy with the proteinase inhibitor saquinavir. 922 47

Sequence analysis of integrated proviruses of human immunodeficiency virus type 1 (HIV-1) which utilize tRNA(His) to initiate reverse transcription [virus derived from pHXB2(His-AC-TGT)] revealed five additional nucleotide substitutions in the U5 and primer binding site (PBS) regions (ATGAC for CCTGT at nucleotides 152, 160, 174, 181, and 200, respectively) (Z. Zhang et al., Virology 226:306-317, 1996). We constructed a mutant proviral genome [pHXB2(His-AC-GAC)] which contained the ATGAC substitutions to test if they represented a necessary adaptation by the virus for use of tRNA(His) to initiate reverse transcription. Viruses from pHXB2(His-AC-TGT) and pHXB2(His-AC-GAC) were infectious. Sequence analysis of the U5 and PBS regions of integrated provirus from a cell culture infected with virus derived from pHXB2(His-AC-TGT) revealed a G-to-A change in CCTGT at nucleotide 181 after limited in vitro culture, suggesting that this nucleotide change represented an adaptation by the virus to efficiently utilize tRNA(His) to initiate reverse transcription. To further address this possibility, we used a specific mutation in reverse transcriptase (RT), a methionine-to-valine change in the highly conserved YMDD amino acid motif of HIV-1 RT (M184V), which has been shown in previous studies to influence the fidelity and activity of the enzyme. The M184V RT mutation was cloned into pHXB2(His-AC-GAC) and pHXB2(His-AC-TGT). Virus derived from pHXB2(His-AC-GAC) with M184V RT had slightly delayed replication compared to the virus from pHXB2(His-AC-GAC) with wild-type RT; in contrast, virus from pHXB2(His-AC-TGT) with M184V RT was severely compromised in replication. Using an endogenous reverse transcription-PCR assay to analyze the reverse transcription of viruses obtained after transfection, we found that viruses derived from pHXB2(His-AC-GAC) with the wildtype RT were slightly faster in the initiation of reverse transcription than viruses with M184V RT. The initiation of reverse transcription was delayed in viruses derived from pHXB2(His-AC-TGT) with wild-type RT and M184V RT compared to viruses derived from pHXB2(His-AC-GAC). Finally, sequence analysis of U5 and PBS regions of proviruses from pHXB2(His-AC-GAC) with wild-type RT revealed considerably more nucleotide substitutions than in viruses derived from pHXB2(His-AC-GAC) containing the M184V mutation in RT after extended in vitro culture. Our studies point to a role for these additional nucleotide substitutions in U5 as an adaptation by the virus to utilize an alternative tRNA to initiate reverse transcription.
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PMID:Nucleotide substitutions within U5 are critical for efficient reverse transcription of human immunodeficiency virus type 1 with a primer binding site complementary to tRNA(His). 926 48

Although the Nef proteins encoded by human immunodeficiency virus type 1 (HIV-1) and simian immuno-deficiency virus (SIV) are known to induce the efficient internalization and degradation of cell surface CD4, it remains unclear whether this process involves a direct interaction between Nef and CD4. Here, we report that CD4 downregulation by HIV-1 and SIV Nef requires distinct but overlapping target sites within the CD4 intracytoplasmic domain. In particular, mutation of a glutamic acid residue located at CD4 residue 405 or of arginine and methionine residues located, respectively, at residue 406 and 407 results in a mutant CD4 protein that is efficiently downregulated by HIV-1 Nef but refractory to downregulation by SIV Nef. However, both HIV-1 and SIV Nef require an isoleucine located at residue 410 and the dileucine motif found at CD4 residues 413 and 414. CD4 downregulation induced by the Nef protein encoded by HIV-2 is shown to require a CD4 target sequence that is similar to, but distinct from, that observed with SIV Nef. These data explain the previous finding that the murine CD4 protein, which has an alanine at residue 405, is refractory to downregulation by SIV, but not HIV-1, Nef (J. L. Foster, S.J. Anderson, A. L. B. Frazier, and J. V. Garcia, Virology 201:373-379, 1994). In addition, these observations provide strong genetic support for the hypothesis that the Nef-mediated downregulation of cell surface CD4 requires a direct Nef-CD4 interaction.
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PMID:Human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef use distinct but overlapping target sites for downregulation of cell surface CD4. 926 98

The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "template grip" have close contacts with the template strand and aid in positioning the template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.
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PMID:Protein-nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double-stranded DNA template-primer. 935 57

Reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) has low fidelity compared with RTs of other retroviruses and cellular DNA polymerases. We and others have previously found that the fidelity of DNA-dependent DNA polymerization (DDDP) of M184V-mutated HIV-1 RT is significantly higher than that of wild-type RT. Viruses containing the M184V substitution are highly resistant to (-)-2'-dideoxy-3'-thiacytidine (3TC) in vitro and in patients treated with 3TC monotherapy. It was of interest to determine the fidelity of RNA-dependent DNA polymerization (RDDP) of M184V RT compared with wild-type because this step occurs first in reverse transcription; errors made during this step may be copied in subsequent polymerization steps. Using an in vitro mispaired primer extension assay, M184V-mutated RT exhibited 3-49-fold decreased frequency of mispair extension compared with wild-type RT. Fidelity differences between M184V and wild-type RT were most marked in extension of A:G (49-fold) and A:C (16-fold) mispairs, with only a marginal (3-fold) decrease in the extension of A:A mispairs. RT containing a methionine to isoleucine (M184I) mutation showed only slight increases in RDDP fidelity compared with wild-type, ranging from 1.5- to 6-fold increases. Of the three RTs tested, wild-type RT was the most error-prone, with mispair extension frequencies ranging from 6.674 x 10(-1) to 7.454 x10(-2).
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PMID:Higher fidelity of RNA-dependent DNA mispair extension by M184V drug-resistant than wild-type reverse transcriptase of human immunodeficiency virus type 1. 935 62

Replication of zidovudine-resistant human immunodeficiency virus type 1 (HIV-1) strains (containing the 41 Met-->Leu and 215 Thr-->Tyr mutations in reverse transcriptase [RT]) was inhibited to a significantly greater extent by the combination of lamivudine and quinoxaline HBY 097 than by either drug alone or even fully suppressed by concomitant HBY 097 and lamivudine administration at relatively low concentrations. The virus recovered after exposure to the drug combinations individually had acquired the 103 Lys-->Arg, 138 Glu-->Lys, 184 Met-->Ile, and 189 Val-->Ile mutations in the genetic zidovudine-resistance background of zidovudine-resistant HIV-1. These mutants retained marked sensitivity to HBY 097. The genotypic zidovudine-resistance mutations were maintained in the mutant virus RT genomes, and the viruses also remained phenotypically resistant to zidovudine. Given the exquisite potency of the combination of lamivudine and HBY 097 in suppressing viral replication, this combination should be further pursued in clinical trials examining treatment of HIV-1-infected persons.
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PMID:Zidovudine-resistant human immunodeficiency virus type 1 strains subcultured in the presence of both lamivudine and quinoxaline HBY 097 retain marked sensitivity to HBY 097 but not to lamivudine. 935 46


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