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 simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described. The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid. A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting. In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C. When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease. Recombinant p24 was purified to near homogeneity by a simple three-step procedure. The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E. coli by the coexpressed protease. The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.
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PMID:A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1. 147 33

The gag and pol genes of the human immunodeficiency virus type 1 (HIV-1) (ref. 1) are translated as two polyproteins, Pr55gag and Pr160gag-pol (refs 2-6), which are subsequently cleaved by the action of a virus-encoded protease into the four structural gag proteins of the virion core (p17, p24, p7 and p6) and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H, and endonuclease). Mutational inactivation of the proteases of HIV-1 and other retroviruses results in immature, non-infectious virions, indicating that exogenous inhibition of the protease may represent an attractive approach to anti-AIDS therapy. Here we demonstrate that synthetic peptide analogues, which are potent inhibitors of purified HIV-1 protease, inhibit the processing of the viral polyproteins in cultures of HIV-1-infected T lymphocytes and attenuate viral infectivity.
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PMID:Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. 168 46

Retroviruses encode proteinases necessary for the proteolytic processing of the viral gag and gag-pol precursor proteins. These enzymes have been shown to be structurally and functionally related to aspartyl proteinases such as pepsin and renin. Cerulenin is a naturally occurring antibiotic, commonly used as an inhibitor of fatty acid synthesis. Cerulenin has been observed to inhibit production of Rous sarcoma virus and murine leukaemia virus by infected cells, possibly by interfering with proteolytic processing of viral precursor proteins. We show here that cerulenin inhibits the action of the HIV-1 proteinase in vitro, using 3 substrates: a synthetic heptapeptide (SQNYPIV) which corresponds to the sequence at the HIV-1 gag p17/p24 junction, a bacterially expressed gag precursor, and purified 66 kDa reverse transcriptase. Inhibition of cleavage by HIV-1 proteinase required preincubation with cerulenin. Cerulenin also inactivates endothiapepsin, a well-characterised fungal aspartyl proteinase, suggesting that the action of cerulenin is a function of the common active site structure of the retroviral and aspartic proteinases. Molecular modelling suggests that cerulenin possesses several of the necessary structural features of an inhibitor of aspartyl proteinases and retroviral proteinases. Although cerulenin itself is cytotoxic and inappropriate for clinical use, it may provide leads for the rational design of inhibitors of the HIV proteinase which could have application in the chemotherapy of AIDS.
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PMID:In vitro inhibition of HIV-1 proteinase by cerulenin. 169 Jan 52

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.
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PMID:A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites. 200 37

Heptapeptide substrates containing a single amino acid substitution at the p2' position of the gag p17-p24 junction (S-Q-N-Y-P-X-V where X = G, A, L, I, F, and W) were compared as substrates for HIV-1 protease. Binding of the Ile-, Leu-, and Ala- containing peptides was about equal although hydrolysis was 20-fold lower for the Ala and Leu peptides compared to Ile. Insertion of Gly or Phe at the p2' position resulted in significantly lower cleavage of the peptide while a Trp-containing peptide was not cleaved. These data suggest that a relatively small hydrophobic amino acid is important for hydrolysis and binding at this site. Structure-activity studies such as those described here may be useful in the design of specific inhibitors for HIV-1 protease.
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PMID:Substitutions at the P2' site of gag p17-p24 affect cleavage efficiency by HIV-1 protease. 218 16

A series of peptide derivatives based on the transition-state mimetic concept has been designed that inhibit the proteinase from the human immunodeficiency virus (HIV). The more active compounds inhibit both HIV-1 and HIV-2 proteinases in the nanomolar range with little effect at 10 micromolar against the structurally related human aspartic proteinases. Proteolytic cleavage of the HIV-1 gag polyprotein (p55) to the viral structural protein p24 was inhibited in chronically infected CEM cells. Antiviral activity was observed in the nanomolar range (with one compound active below 10 nanomolar) in three different cell systems, as assessed by p24 antigen and syncytium formation. Cytotoxicity was not detected at 10 and 5 micromolar in C8166 and JM cells, respectively, indicating a high therapeutic index for this new class of HIV proteinase inhibitors.
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PMID:Rational design of peptide-based HIV proteinase inhibitors. 218 54

Recombinant vaccinia viruses that contained regions of the gag-pol open reading frames of human immunodeficiency virus type 1 (HIV-1) were constructed. Cells infected with recombinants containing both gag and protease genes expressed and processed HIV gag antigens efficiently. Processing was much reduced in cells infected with recombinants containing only gag, but not the protease gene. However, significant amounts of p41 were produced by protease-defective recombinants. This protein was immunoreactive with p24-specific monoclonal antibodies and was produced in a truncated form by a recombinant containing a 3' deletion in the p15 coding region of gag ORF. These results indicate that p41 could represent an alternative gag precursor with N-terminal sequences derived from p24 and C-terminal from p15. Ultrastructural analysis of recombinant-infected cells revealed that the gag antigens expressed were assembled into retrovirus-like particles and were secreted into culture medium. This assembly process was not dependent on HIV protease function, because immature core particles were produced by recombinants lacking HIV-1 protease functions. Immunization of mice and chimpanzees with vaccinia-HIVgag recombinant viruses generated both antibody and cell-mediated immune responses to HIV gag antigens. These recombinants are therefore useful not only for studying HIV virion processing and assembly, but also for designing immunogens for the prophylaxis and immunotherapy against AIDS.
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PMID:Processing, assembly, and immunogenicity of human immunodeficiency virus core antigens expressed by recombinant vaccinia virus. 221 27

The ability of two novel synthetic compounds to inhibit the HIV protease-mediated processing of HIV-1 precursor polyproteins was investigated in an in vitro gag-protease mixed lysate assay system and in an assay using recombinant baculoviruses engineered to express the HIV-1 gag and pol genes in cultured insect cells. With the in vitro mixed lysate assay we have shown that both compounds at 1 microM can completely inhibit the HIV-1 and HIV-2 protease-mediated release of p24 from the HIV-1 gag precursor at pH 5.5 and pH 7.0. In the intracellular baculovirus system these compounds were shown to inhibit the protease-mediated maturation of gag and also the excision of the protease moiety from its precursor.
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PMID:Effect of two novel inhibitors of the human immunodeficiency virus protease on the maturation of the HIV gag and gag-pol polyproteins. 221 37

A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product [tyrosyl-3,5-3H]Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.
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PMID:A radiometric assay for HIV-1 protease. 222 92

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


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