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)

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

Replacement of the putative active site Asp residue of cloned HIV-1 protease with Ala yields a molecule incapable of autocatalytic processing. Similarly, protease/reverse transcriptase and protease/reverse transcriptase/endonuclease polyproteins containing the same mutation accumulate as enzymatically inert polyproteins. Introduction of a second, wild-type, copy of protease in trans alleviates this defect, leading in the case of individually cloned protease to cleavage of the mutant protein, and with the polyprotein mutants to release of the reverse transcriptase and endonuclease polypeptides, the former of which recover enzymatic activity. In related experiments, a similar inhibition and trans-complementation of a genetically engineered gag--protease fusion protein was observed.
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PMID:Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation. 246 Dec 97

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

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.
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PMID:Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases. 264 5

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

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E. coli. A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17-p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/micrograms of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.
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PMID:Chemical synthesis and expression of the HIV-1 protease gene in E. coli. 264 85

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

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.
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PMID:Substitution mutations of the highly conserved arginine 87 of HIV-1 protease result in loss of proteolytic activity. 267 52

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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