Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Bovine pancreatic ribonuclease A (RNase) contains two bonds, Met29-Met30 and Tyr92-Pro93 which are representative of sites in the human immunodeficiency virus-1 (HIV-1) gag polyprotein precursors that are cleaved by the HIV-1 protease during viral maturation. Nevertheless, neither native nor performic acid-oxidized RNase is a substrate for the protease. However, RNase derivatives obtained by reduction and S-alkylation with iodoacetate or iodoacetamide undergo cleavage by the HIV-1 protease at a single site, Ala109-alkyl-Cys110, that is distinct from either of the two predicted bonds mentioned above. The neutral carboxyamido-methylcysteinyl derivative is cleaved 8 times faster than that containing the negatively charged carboxy-methyl substituent at P1'. Succinylation of these S-alkylated RNase derivatives creates a second site of cleavage by the protease between succinyl-Lys7 and Phe8. Thus, the pattern of cleavage of denatured RNase by the HIV-1 protease can be manipulated by chemical derivatization of the substrate, and the new sites of hydrolysis revealed by these studies add to our understanding of the specificity of this important enzyme.
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PMID:Ribonuclease A as a substrate of the protease from human immunodeficiency virus-1. 225 31

A synthetic peptidemimetic substrate of the human immunodeficiency virus 1 (HIV-1) protease with a nonhydrolyzable pseudodipeptidyl insert at the protease cleavage site was prepared. The peptide U-81749 inhibited recombinant HIV-1 protease in vitro (inhibition constant Ki of 70 nanomolar) and HIV-1 replication in human peripheral blood lymphocytes (inhibitory concentration IC50 of 0.1 to 1 micromolar). Moreover, 10 micromolar concentrations of U-81749 significantly inhibited proteolysis of the HIV-1 gag polyprotein (p55) to the mature viral structural proteins p24 and p17 in cells infected with a recombinant vaccinia virus expressing the HIV-1 gag-pol genes. The HIV-1 like particles released from inhibitor-treated cells contained almost exclusively p55 and other gag precursors, but not p24. Incubation of HIV-like particles recovered from drug-treated cultures in drug-free medium indicated that inhibition of p55 proteolysis was at least partially reversible, suggesting that U-81749 was present within the particles.
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PMID:A synthetic HIV-1 protease inhibitor with antiviral activity arrests HIV-like particle maturation. 240 86

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)/ribonuclease H has been expressed to high levels in Escherichia coli from a recombinant plasmid constructed using the polymerase chain reaction (PCR) for in vitro mutagenesis. Translational initiation and termination codons were introduced by the PCR at points corresponding to sites of cleavage of the RT from the gag-pol precursor polyprotein by the HIV-1 protease; the HIV-1 protease is not expressed from this construct. Most of the RT coding sequences derived from PCR were exchanged for a DNA fragment cloned by standard methods to minimize the possibility that an unwanted mutation was introduced during the in vitro amplification. The RT is expressed in bacteria from this plasmid as 66 and 51 kDa proteins, has both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities, and is indistinguishable from native HIV-1 RT in electrophoretic mobility and immunoreactivity. Peptide sequencing of the amino terminus of the HIV-1 RT purified from bacterial lysates is also presented. A novel activity gel assay was used to confirm that only the 66 kd protein catalyzes the RNase H reaction; this assay will simplify analysis of this catalytic activity. This HIV-1 RT expression plasmid is of interest because of the high level of expression in bacteria and the demonstrated RNase H activity of the enzyme. This plasmid will be distributed for research purposes through the NIH AIDS Repository and will facilitate enzymologic, structural, and immunologic evaluation of reverse transcription and its chemotherapeutic inhibition.
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PMID:HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. 247 33

The human immunodeficiency virus (HIV-1) encodes a protease that is essential for viral replication and is a member of the aspartic protease family. The recently determined three-dimensional structure of the related protease from Rous sarcoma virus has been used to model the smaller HIV-1 dimer. The active site has been analyzed by comparison to the structure of the aspartic protease, rhizopuspepsin, complexed with a peptide inhibitor. The HIV-1 protease is predicted to interact with seven residues of the protein substrate. This information can be used to design protease inhibitors and possible antiviral drugs.
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PMID:Molecular modeling of the HIV-1 protease and its substrate binding site. 253 31

Retroviral capsid proteins and replication enzymes are synthesized as polyproteins that are proteolytically processed to the mature products by a virus-encoded proteinase. We have purified the proteinase of human immunodeficiency virus (HIV), expressed in Escherichia coli, to approximately 90% purity. The purified enzyme at a concentration of approximately 20 nM gave rapid, efficient, and specific cleavage of an in vitro synthesized gag precursor protein. Purified HIV proteinase also induced specific cleavage of five decapeptide substrates whose amino acid sequences corresponded to cleavage sites in the HIV polyprotein but not of a peptide corresponding to a cleavage site in another retrovirus. Competition experiments with different peptides allowed a ranking of cleavage sites. Inhibition studies indicated that the HIV proteinase was inhibited by pepstatin A with an IC50 of 0.7 microM.
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PMID:Activity of purified biosynthetic proteinase of human immunodeficiency virus on natural substrates and synthetic peptides. 264 44

Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55gag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease.
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PMID:Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease. 264 84

It is generally believed that the gag gene product of human immunodeficiency virus type 1 (HIV-1) is processed into several core proteins by a virus-specific protease. We used deletion mutation analysis to study the role of HIV-specific protease in the processing of core proteins and its requirement for viral infectivity. Several mutant genomes with deletions in the protease gene were constructed. A mammalian cell line, COS-M6, transfected with the wild-type viral genome was shown to produce virions containing processed core proteins, while COS-M6 cells transfected with two mutated genomes could express only the core protein precursor, Pr56gag. The wild-type transfectant produced infectious virus; both transfectants expressing the mutated genomes also produced virions, and one of them still retained reverse transcriptase activity. However, the mutant viral particles were devoid of infectivity. Virions with a distinct central core and an electron-dense nucleoid budded out from the plasma membrane of COS-M6 cells transfected with the wild-type genome. In contrast, noninfectious virions that budded either into cytoplasmic vacuoles or out from the plasma membrane of COS-M6 cells transfected with mutant genomes contained ring-shaped nucleoids. These results indicate that the HIV-1 protease plays a role not only in the maturation of the core proteins but also in the assembly of the virus and thus is required for viral infectivity.
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PMID:Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity. 265 99

Three groups with different routes of human immunodeficiency virus type 1 (HIV-1) transmission (homosexual men, hemophiliacs, and children) were studied for serum antibodies to a recombinant form of the HIV-1 protease using an enzyme-linked immunoassay. At 1 year after seroconversion, defined as the moment antibodies to HIV-1 proteins were first detected, 56% (34/61) of the homosexual men had antibodies to protease, and 2 years after seroconversion this percentage was 63% (24/38). Within this 2-year period these antibodies were no longer detected in 16% (9/56). A similar pattern was observed in 20 hemophiliacs who seroconverted after exposure to HIV-1-contaminated blood products. We found that 63% (160/255) of homosexual men in Centers for Disease Control stage II or III, 60% (9/15) of patients with acquired immunodeficiency syndrome (AIDS)-related complex, and 36% (14/39) of patients with AIDS had antibodies to protease. In 255 homosexual men in Centers for Disease Control stage II or III, antibodies to protease were significantly more frequently found in samples lacking HIV-1 antigen (P less than 0.001) and possessing antibodies to HIV-1 core proteins (P less than 0.001). Twenty-four persons who developed AIDS were studied longitudinally: 58% (14/24) had antibodies to protease 1 year before developing symptoms; 29% (7/24) showed a decline and 29% (7/24) showed a loss of antibodies to protease at the onset of symptoms. Within a group of 47 HIV-1-infected children, 90% (18/20) with a stable disease course were persistently protease antibody positive, versus 4 of 27 children (15%) with an unstable disease course (P = 0.0001). These data indicate that HIV-1 protease is expressed and antigenic in most HIV-1-infected individuals and that a decline or absence of antibodies to protease is strongly associated with unstable disease in children and AIDS in adults.
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PMID:Antibody response to human immunodeficiency virus type 1 protease according to risk group and disease stage. 267 Oct 17

Inhibitors of the protease from human immunodeficiency virus 1 (HIV-1) were designed, synthesized, and kinetically characterized. Analogues of a heptapeptide substrate of HIV-1 protease with sequence similar to the p17-p24 cleavage site in the natural substrate, Pr55gag, were synthesized in which the scissile dipeptide bond was replaced with bonds from six categories of stable mimics of an aspartic proteolysis transition state or intermediate. These mimics included an analogue of statine, hydroxyethylene isosteres, two categories of phosphinic acids, a reduced amide isostere, and an alpha,alpha-difluoroketone. The resulting peptide analogues were linear competitive inhibitors of purified recombinant HIV-1 protease with inhibition constants ranging from 18 nM to 40 microM depending on the type of inhibitor. A truncated inhibitor, an analogue of a hexapeptide, retained full inhibitory potency. The most potent inhibitors, containing the hydroxyethylene isostere, effectively blocked the proteolytic processing of a recombinant form of Pr55gag by HIV-1 protease in a cell-free assay.
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PMID:Inhibition of human immunodeficiency virus 1 protease in vitro: rational design of substrate analogue inhibitors. 269 72

The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides. Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS. In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease. Expression of the protease in E. coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease. Extracts of induced cultures of E. coli harboring a protease-containing plasmid served as the source of protease activity. The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24. Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction. The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction.
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PMID:Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease. 269 58


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