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)

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.
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PMID:The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein. 983 4

HIV-1 reverse transcriptase (RT) is a key enzyme involved in the replication of the virus and is a potential target for therapeutic intervention following infection. Several drugs that inhibit the enzyme from acting have been discovered. These include nucleoside-analogue inhibitors such as AZT (zidovudine), ddI and ddC, and non-nucleoside inhibitors such as nevirapine and delavirdine. All of them, however have been found to be of limited clinical utility because the RT becomes rapidly resistant to them on account of point mutations in the enzyme. One way to partly overcome this limitation is to design an inhibitor that has interactions mainly with the backbone and the conserved residues of RT. Using a rational drug-design approach based on the high resolution X-ray crystal structure of the RT-nevirapine complex (1), and the specific design principles of peptides containing dehydro-Alanine (deltaAla) generated by our theoretical calculations, we present here the design of a peptide inhibitor of RT. Energy minimization and molecular modeling of the interaction of the designed pentapeptide with the nevirapine-binding site indicate that the inhibitor has 60% of its interactions with the conserved regions of RT as compared to 30% in the case of nevirapine, thus making it much less sensitive to mutations in the enzyme.
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PMID:A peptide inhibitor of HIV-1 reverse transcriptase using alpha,beta-dehydro residues: a structure-based computer model. 983 73

Crystallographic characterization of a ternary complex containing a monomeric gp120 core, parts of CD4, and a mAb, revealed a region that bridges the inner and outer domains of gp120. In a related genetic study, several residues conserved among primate lentiviruses were found to play important roles in CC-chemokine receptor 5 (CCR5) coreceptor utilization, and all but one were mapped to the bridging domain. To reconcile this finding with previous reports that the hypervariable region 3 (V3) of gp120 plays an important role in chemokine coreceptor utilization, elucidating the roles of various V3 residues in this critical part of the HIV type 1 (HIV-1) life cycle is essential. Alanine-scanning mutagenesis was carried out to identify V3 residues critical for CCR5 utilization. Our findings demonstrated that several residues in V3 were critical to CCR5 utilization. Furthermore, these residues included not only those conserved across HIV-1 subtypes, but also those that varied among HIV-1 subtypes. Although the highly conserved V3 residues may represent unique targets for antiviral designs, the involvement of variable residues raises the possibility that antigenic variation in the coreceptor binding domain could further complicate HIV-1 vaccine design.
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PMID:Hypervariable region 3 residues of HIV type 1 gp120 involved in CCR5 coreceptor utilization: therapeutic and prophylactic implications. 1020 Mar 1

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.
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PMID:Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity. 1038 87

CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.
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PMID:Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein. 1057 39

We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, we have shown that the C-terminal deletion mutants of Sam68 act with a transdominant negative phenotype in HIV replication. Previously, an Arginine429 mutation within the C-terminal domain of Sam68 has been reported to be critical for the localization of Sam68 in the nucleus. However, these studies were done in the context of truncated protein in which C-terminal amino acids 420 - 443 of Sam68 were fused to GFP. In contrast, we now report that the full length Sam68 protein having the same mutation (Arginine429-->Alanine) is completely localized in the nucleus while another Sam68 (Proline439-->Arginine) mutant is found in the cytoplasm. The localization of these Sam68 mutant proteins also correlates with their function in RRE-mediated reporter gene expression, i.e. Sam68 mutant protein that is localized in the cytoplasm failed to enhance RRE-mediated transactivation. Furthermore, we demonstrate that Sam68 P439-->R inhibited the transactivation of RRE-mediated gene expression by both wild type Sam68 and Rev. These results indicate that the proline residue at position 439 unlike arginine at position 429, may play a critical role in targeting Sam68 protein to nucleus. We propose that these negative dominant mutants of Sam68 may have potential as anti-viral agents to combat AIDS.
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PMID:A single point mutation in the nuclear localization domain of Sam68 blocks the Rev/RRE-mediated transactivation. 1087 64

Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.
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PMID:Crystal structure of recombinant native SDF-1alpha with additional mutagenesis studies: an attempt at a more comprehensive interpretation of accumulated structure-activity relationship data. 1095 12

HIV-1 encodes an aspartic protease, an enzyme crucial to viral maturation and infectivity. It is responsible for the cleavage of various protein precursors into viral proteins. Inhibition of this enzyme prevents the formation of mature, infective viral particles and therefore, it is a potential target for therapeutic intervention following infection. Several drugs that inhibit the action of this enzyme have been discovered. These include peptidomimetic inhibitors such as ABT-538 and saquinavir, and structure based inhibitors such as indinavir and nelfinavir. Several of these have been tested in human clinical trials and have demonstrated significant reduction in viral load. However, most of them have been found to be of limited clinical utility because of their poor pharmacological properties and also because the viral protease becomes rapidly resistant to these drugs on account of mutations in the enzyme. One way to overcome these limitations is to design an inhibitor that interacts mainly with the conserved residues of HIV-1 protease. By a rational drug design approach based on the high resolution X-ray crystal structure of the HIV-1 protease with--MVT 101 (a substrate based inhibitor) and the specific design principles of peptides containing dehydro-Alanine (delta Ala) derived from our earlier studies, we have designed a tetrapeptide with the sequence: NH2-Thr-delta Ala-delta Ala-Gln-COOH. Energy minimization and molecular modelling of the interaction of the designed tetrapeptide with the inhibitor binding site indicate that the inhibitor is in an extended conformation and makes excessive contacts with the viral enzyme at the interface between the protein subunits. The designed inhibitor has 33% of its interaction with the conserved region of HIV-1 protease which is of the same order as that of MVT 101 with the enzyme.
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PMID:A peptide inhibitor of HIV-1 protease using alpha, beta- dehydro residues: a structure based computer model. 1156 39

Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.
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PMID:Structural and functional analysis of interhelical interactions in the human immunodeficiency virus type 1 gp41 envelope glycoprotein by alanine-scanning mutagenesis. 1160 54

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (gp120-gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. Formation of a stable trimer-of-hairpins structure in the gp41 ectodomain brings the two membranes into proximity, leading to membrane fusion. The core of this hairpin structure is a six-helix bundle in which three carboxyl-terminal outer helices pack against an inner trimeric coiled coil. Here we investigate the role of these conserved interhelical interactions on the structure and function of both the envelope glycoprotein and the gp41 core. We have replaced each of the eight amino acids at the buried face of the carboxyl-terminal helix with a representative amino acid, alanine. Structural and physicochemical characterization of the alanine mutants shows that hydrophobic interactions are a dominant factor in the stabilization of the six-helix bundle. Alanine substitutions at the Trp628, Trp631, Ile635, and Ile642 residues also affected envelope processing and/or gp120-gp41 association and abrogated the ability of the envelope glycoprotein to mediate cell-cell fusion. These results suggest that the amino-terminal region of the gp41 outer-layer alpha-helix plays a key role in the sequence of events associated with HIV-1 entry and have implications for the development of antibodies and small-molecule inhibitors of this conserved element.
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PMID:Interhelical interactions in the gp41 core: implications for activation of HIV-1 membrane fusion. 1204 59


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