Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
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PMID:Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase. 204 56

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for processing viral polyproteins which contain the enzymes and structural proteins required for the infectious virus. It was previously found that cupric chloride, in the presence of dithiothreitol or ascorbic acid, could inhibit the HIV-1 protease. It was suggested that a Cu1+ chelate was the moiety responsible for inhibition of the protease. This hypothesis has now been investigated directly by utilizing the stable Cu1+ chelate, bathocuproine disulfonic acid Cu1+ (BCDS-Cu1+). BCDS-Cu1+ inhibited the HIV-1 wild type protease as well as a mutant HIV-1 protease lacking cysteines. BCDS-Cu1+ was a competitive inhibitor of the mutant HIV-1 protease with an apparent Ki of 1 microM. Replication of HIV-1 in human lymphocytes and the cytotoxic effect of HIV-1 in CEM cells was inhibited by micromolar BCDS-Cu1+. Inhibition of the protease and of HIV replication by BCDS-Cu1+ was dependent on the presence of Cu1+ as BCDS alone was ineffective. EDTA blocked the inhibition of the protease by Cu1+ but was unable to block inhibition of the protease by BCDS-Cu1+, indicating that the Cu1+ complex was the inhibitory agent. The apparent IC50 for BCDS-Cu1+ on the inhibition of replication by primary isolates of HIV-1 was 5 microM. However, BCDS-Cu1+ did not affect polyprotein processing in an H9 cell line chronically infected with HIV-1, indicating that BCDS-Cu1+ acts by yet another mechanism to block HIV infection. Other possible targets for BCDS-Cu1+ include inhibition of viral adsorption and/or inhibition of the HIV-1 integrase.
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PMID:Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+. 757 66

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.
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PMID:Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles. 768 10

Several new analogues of the novel anti-HIV agent cosalane have been synthesized and evaluated as inhibitors of HIV-1 integrase and protease, HIV-1 replication, HIV-1 and HIV-2 cytopathicity, HIV-1- and HIV-2-mediated syncytium formation, and cytopathicity of a variety of human pathogenic viruses. The congeners displayed enhanced potencies relative to cosalane itself as inhibitors of HIV-1 integrase and protease. The two most potent analogues against HIV-1 integrase displayed IC50 values of 2.2 microM, while the three most potent compounds against HIV-1 protease had IC50 values in the 0.35-0.39 microM range. In addition to its activity against HIV-1 and HIV-2 cytopathicity, cosalane inhibited the cytopathic effects of herpes simplex virus-1, herpes simplex virus-2, and human cytomegalovirus at concentrations that were well below the cytotoxic concentrations. Potentially useful antiviral activities were also revealed for some of the new cosalane congeners against influenza virus, Junin virus, and Tacaribe virus.
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PMID:Cosalane analogues with enhanced potencies as inhibitors of HIV-1 protease and integrase. 785 37

Upon the binding of a synthetic nonapeptide substrate, the catalytically active dimeric form of HIV proteinase is strongly stabilized against dissociation into inactive subunits. The dissociation of the ternary Michaelis complex into protein monomers is immeasurably low (apparent dissociation constant in the picomolar range), while the dimer-to-monomer equilibrium dissociation constant at pH 4.7 and at ionic strength 1.0 M is 30.4 +/- 1.6 nM. Consequently, the apparent activity of HIV proteinase depends on the order in which the enzyme and the substrate are added to in vitro assays. Substrate-induced stabilization should be carefully considered in designing kinetic studies of all dissociative retroviral enzymes, the proteinase, the integrase, and the reverse transcriptase.
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PMID:Stabilization of HIV proteinase dimer by bound substrate. 833 44

A novel series of omega-aminoalkanoic acid derivatives of betulinic acid were synthesized and evaluated for their activity against human immunodeficiency virus (HIV). The anti-HIV-1 activity of several members of this new series was found to be in the nanomolar range in CEM 4 and MT-4 cell cultures. The optimization of the omega-aminoalkanoic acid side chain is described. The presence of an amide function within the side chain was found important for optimal activity. RPR 103611 (14g), a statine derivative, was found to be inactive against HIV-1 protease, reverse transcriptase, and integrase as well as on gp120/CD4 binding. "Time of addition" experiments suggested interaction with an early step of HIV-1 replication. As syncytium formation, but not virus-cell binding, seems to be affected, betulinic acid derivatives are assumed to interact with the postbinding virus-cell fusion process.
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PMID:Betulinic acid derivatives: a new class of specific inhibitors of human immunodeficiency virus type 1 entry. 867 42

Acute HIV-1 infection of H9 and C8166 cultures has been shown to be suppressed by certain flavonoids, and evidence for inhibition of HIV-1 protease, integrase, and reverse transcriptase by flavonoids also exists. The present aim was to determine whether flavonoids inhibit HIV-1 activation in models of latent infection. By screening flavonoids from six different classes, three structurally related compounds (chrysin, acacetin, and apigenin) were identified that inhibited HIV expression in TNF-alpha-treated OM-10.1 cultures. The three compounds had favorable potencies against HIV activation in relation to their growth inhibitory effects (therapeutic index 5-10). Chrysin also inhibited HIV expression in response to PMA in OM-10.1 cells, in ACH-2 cells stimulated with either TNF-alpha or PMA, and in 8E5 cultures. Furthermore, return to viral latency in OM-10.1 cells previously exposed to TNF-alpha occurred over a shorter time interval when chrysin was added. The inhibition of HIV activation was not dependent on preincubation with flavonoids relative to TNF, and was characterized by a lack of HIV RNA accumulation by Northern analysis. Gel-shift experiments revealed that NF-kappa B activation after TNF-alpha treatment was not inhibited by these agents, suggesting that some other critical factor(s) needed for viral transcription was being affected. These findings indicate that flavonoids inhibit HIV-1 activation via a novel mechanism, and that these agents are potential candidates for therapeutic strategies aimed at maintaining a cellular state of HIV-1 latency.
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PMID:Inhibition of HIV activation in latently infected cells by flavonoid compounds. 882 17

A temperature-sensitive (ts) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutant was generated by charged-cluster-to-alanine mutagenesis. The mutant virus, containing three charged residues within the RT finger domain changed to alanine (K64A, K66A, and D67A), replicated normally at 34.5 but not 39.5 degrees C. Quantitating virus particle production by p24 antigen capture or virion-associated RT activity and virus infectivity by the MAGI cell assay, we found that (i) mutant virions produced at the permissive temperature were indistinguishable from wild-type virus in assays performed at the nonpermissive temperature, suggesting that the ts mutation did not impair early steps in the virus replication cycle and that the mutant RT enzyme was not ts; and (ii) virus particle production in cells transfected with the ts mutant at the nonpermissive temperature was comparable to that of wild-type virus. However, the particle-associated RT activity and infectivity of mutant virions produced at the nonpermissive temperature were greatly reduced when assays were conducted at the permissive temperature. These results are consistent with an irreversible ts event affecting RT that occurs during virus particle production. Radioimmunoprecipitation analyses revealed that both p66 and p51 RT subunits were absent from mutant virions generated at 39.5 degrees C. The presence of normal levels of HIV-1 integrase in mutant particles produced at the nonpermissive temperature was inconsistent with defective Gag-Pol synthesis or Gag-Pol incorporation into progeny virions. Furthermore, wild-type levels of the mutant Pr160(gag-pol) were detected in virions produced at the nonpermissive temperature when the HIV-1 protease was inactivated by site-specific mutagenesis. Taken together, these results are most consistent with a ts defect affecting the degradation or aberrant processing of the mutated RT during its processing/maturation within nascent particles.
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PMID:Construction and characterization of a temperature-sensitive human immunodeficiency virus type 1 reverse transcriptase mutant. 949 59

A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and coexpression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permitting virion incorporation of the chimeric protein when expressed, in trans, with other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for integration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (double copy of WF domain) complementation is not improved markedly by incorporation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clones, both of which bear an intervening protease cleavage site, appear comparable. Western blotting analyses using virions isolated through sucrose cushions demonstrate clearly the incorporation of the dWF-IN fusion protein into Vpr containing HIV-1 particles but not in Vpr-deficient virions. Additional Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of virion incorporation of Vpr-IN and dWF-IN chimeras appears approximately comparable by Western blotting analysis. The ability of dWF-IN to complement IN-defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-PR-IN indicates that dWF-IN retains the full complement of functions necessary for integration of proviral DNA and is likely due to the benign nature of this small domain at the amino-terminus of IN.
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PMID:A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions. 1004 23

Poly(1-methyl-6-thioinosinic acid), or PMTI, is a single-stranded polyribonucleotide and is the first homopolyribonucleotide devoid of Watson-Crick hydrogen bonding sites to show potent human immunodeficiency virus (HIV) inhibition. PMTI was found to be active when evaluated against a variety of low passage clinical HIV isolates in fresh human peripheral blood cells, including T cell-tropic and monocyte-macrophage-tropic viruses, syncytium-inducing and non-syncytium-inducing viruses and viruses representative of the various HIV-1 clades (A through F). The compound was active against HIV-2, all nucleoside and non-nucleoside reverse transcriptase (RT) inhibitor drug-resistant virus isolates tested and interacted with AZT or ddl to synergistically inhibit HIV infection. In biochemical inhibition assays, PMTI was determined to be a potent inhibitor of HIV-1 and HIV-2 RT, including RTs with mutations that engender resistance to nucleoside and non-nucleoside RT inhibitors. PMTI inhibited both the polymerase and RNase H activities of HIV RT. PMTI did not inhibit HIV-1 protease or integrase. Cell-based mechanism of action assays indicated that PMTI also interfered with early events in the entry of HIV into target cells. Furthermore, PMTI inhibited the fusion of gp120-expressing and CD4-expressing cells, but at concentrations approximately 1 log10 greater than those that inhibited virus entry. These results suggest that the homopolyribonucleotide PMTI blocks HIV replication in human cells at its earliest stages by multiple mechanisms, inhibition of virus entry and inhibition of RT.
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PMID:PMTI, a broadly active unusual single-stranded polyribonucleotide, inhibits human immunodeficiency virus replication by multiple mechanisms. 1007 76


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