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)

The human immunodeficiency virus type 1 (HIV-1) Pr55(gag) gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55(gag) is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55(gag) maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540-8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55(gag) in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55(gag) was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55(gag) was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the "trigger" for a myristoyl switch mechanism that modulates the membrane associations of Pr55(gag) and p17MA in virions and membranes.
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PMID:Human immunodeficiency virus type 1 protease triggers a myristoyl switch that modulates membrane binding of Pr55(gag) and p17MA. 997 69

A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1(SF2) p55(Gag) were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55(Gag) protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55(Gag) and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gag protease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8(+) cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.
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PMID:Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene. 1068 77

We analyzed plasma HIV-1 from 27 antiretroviral drug-naive Ugandan adults. Previous subtype analysis of env and gag sequences from these samples identified subtypes A, C, D, and recombinant HIV-1. Sequences of HIV-1 protease and reverse transcriptase (RT) were obtained with a commercial HIV-1 genotyping system. Subtypes based on protease sequences differed from gag subtypes for 5 of 27 samples, demonstrating a high rate of recombination between the gag and pol regions. Protease and RT sequences were analyzed for the presence of amino acid polymorphisms at positions that are sites of previously characterized drug resistance mutations. At those sites, frequent polymorphisms were detected at positions 36 and 69 in protease and positions 179, 211, and 214 in RT. Subtype-specific amino acid motifs were identified in protease. Most of the subtype A sequences had the amino acids DKKM at positions 35, 57, 69, and 89, whereas most subtype D sequences had the amino acids ERHL at those positions. Detection of those polymorphisms may provide a useful approach for rapid identification of subtype A and D isolates in Uganda. This analysis significantly increases the number of Ugandan protease and RT sequences characterized to date and demonstrates successful use of a commercial HIV-1 genotyping system for analysis of diverse non-B HIV-1 subtypes.
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PMID:Analysis of HIV type 1 protease and reverse transcriptase in antiretroviral drug-naive Ugandan adults. 1082 87

Six boronated tetrapeptides with the carboxy moiety of phenylalanine replaced by dihydroxyboron were synthesized, and their activities against human immunodeficiency virus 1 (HIV-1) protease subsequently investigated. The sequences of these peptides were derived from HIV-1 protease substrates, which included the C-terminal part of the scissile bond (Phe-Pro) within the gag-pol polyprotein. Enzymatic studies showed that these compounds were competitive inhibitors of HIV-1 protease with K(i) values ranging from 5 to 18 microM when experiments were performed at high enzyme concentrations (above 5 x 10(-8) M); however, at low protease concentrations inhibition was due in part to an increase of the association constants of the protease subunits. Ac-Thr-Leu-Asn-PheB inhibited HIV-1 protease with a K(i) of 5 microM, whereas the non-boronated parental compound was inactive at concentrations up to 400 microM, which indicates the significance of boronation in enzyme inhibition. The boronated tetrapeptides were inhibitory to an HIV-1 protease variant that is resistant to several HIV-1 protease inhibitors. Finally, fluorescence analysis showed that the interactions between the boronated peptide Ac-Thr-Leu-Asn-PheB and HIV-1 protease resulted in a rapid decrease of fluorescence emission at 360 nm, which suggests the formation of a compound/enzyme complex. Boronated peptides may provide useful reagents for studying protease biochemistry and yield valuable information toward the development of protease dimerization inhibitors.
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PMID:Inhibition of HIV-1 protease by a boron-modified polypeptide. 1097 1

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 microM. Their activities against HIV-1 protease (K(i) 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC(50) 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC(50) 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 A (1) and 1.85 A (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.
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PMID:Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease. 1100 4

HIV-derived vectors are of potential clinical relevance due to their ability to transduce nondividing cells in vitro and in vivo. However, the generation of cell lines stably and reproducibly expressing high amounts of defined subviral particles, capable of packaging and transducing HIV-derived vectors, has been hampered by the cytotoxicity of some of the required gene products, in particular of the HIV-1 protease. The successful use of regulatable gene expression systems to overcome this problem requires that the remaining basally expressed gene product activity is below the threshold for cytotoxicity. To try to achieve this, we have consecutively introduced appropriate plasmids, encoding HIV rev and HIV gag/pol gene products, each under the control of separate ecdysone-inducible promoters, into human 293 cells. Using a protocol in which a specific HIV protease inhibitor, Saquinavir, was continuously present in the culture medium during selection, we could generate stable cell lines inducibly expressing high amounts of subviral particles. A cell line, termed 293-Rev/Gag/Pol(i), which has been characterized in more detail, inducibly releases, within 48 h postinduction, high amounts of HIV Gag/Pol particles (about 10 microg CA/ml). These HIV Gag/Pol particles can package and transduce third-generation HIV vectors to high titers. Thus, in addition to other applications, the 293-Rev/Gag/Pol(i) cell line represents a "founder" packaging cell line which, depending on the requirement, can be further modified to include specific transgene-encoding vector and targeting glycoprotein genes.
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PMID:Generation of a flexible cell line with regulatable, high-level expression of HIV Gag/Pol particles capable of packaging HIV-derived vectors. 1131 23

Genotype alterations of HIV-1 protease, reverse transcriptase, cleavage sites p7/p1 and p1/p6, as well as p6(gag) and transframe protein p6* were studied in an observational cohort of 42 individuals who received antiretroviral therapy consisting of saquinavir, ritonavir, and two nucleoside analogs. In a multivariate logistic regression analysis, the prior protease inhibitor experience (odds ratio, 6.20; 95% CI, 1.22-31.38) and the presence of primary protease mutations (odds ratio, 9.99; 95% CI, 1.05-94.72) were independently associated with virological failure. Moreover, a trend was observed in that individuals with N-terminal amino acid insertions in the proline-rich motif of the p6(gag) protein were less likely to experience virological failure (OR, 0.17; 95% CI, 0.02-1.35; p = 0.09). In contrast, the presence of secondary protease, reverse transcriptase, or cleavage site mutations was not independently associated with treatment failure. However, mutations at cleavage site p7/p1 (p = 0.01) and C-terminal p6* mutations (p = 0.02) were both associated with primary protease mutations. In conclusion, the presence of primary protease mutations was the most important predictor of the subsequent virological response. Moreover, there is some evidence that insertions in the proline-rich area of the p6(gag) protein may affect the virological response. The relationship between mutations of cleavage sites or C-terminal p6* residues and protease mutations suggests that these alterations may serve a compensatory role, increasing viral fitness.
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PMID:Impact of HIV type 1 protease, reverse transcriptase, cleavage site, and p6 mutations on the virological response to quadruple therapy with saquinavir, ritonavir, and two nucleoside analogs. 1135 Jun 62

HIV-1 protease is a major drug target against AIDS as it permits viral maturation by processing the gag and pol polyproteins of the virus. The cleavage sites in these polyproteins do not have obvious sequence homology or a binding motif and the specificity of the protease is not easily determined. We used various threading approaches, together with the crystal structures of substrate complexes which served as template structures, to study the substrate specificity of HIV-1 protease with the aim of obtaining a better differentiation between binding and nonbinding sequences. The predictions from threading improved when distance-dependent interaction energy functions were used instead of contact matrices. To rank the peptides and properly account for the peptide's conformation in the total energy, the results from using short-range potentials on multiple template structures were averaged. Finally, a dynamic threading approach is introduced which is potentially useful for cases when there is only one template structure available. The conformational energy of the peptide-especially the term accounting for the side chains-was found to be important in differentiating between binding and nonbinding sequences. Hence, the substrate specificity, and thus the ability of the virus to mature, is affected by the compatibility of the substrate peptide to fit within the limited conformational space of the active site groove.
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PMID:Structure-based prediction of potential binding and nonbinding peptides to HIV-1 protease. 1288 33

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag-pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.
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PMID:Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation. 1518 48

Site-specific proteolysis is essential in many fundamental cellular and viral processes. It has been previously shown that the Escherichia coli beta-galactosidase can be useful for the high-throughput screening of human immunodeficiency virus type 1 protease inhibitors. Here, by using crystallographic and functional data of the bacterial enzyme, we have identified a new accommodation site between amino acids 581 and 582, in a solvent-exposed and flexible beta-turn of domain III. The placement of the model peptide reproducing the matrix-capsid (p17/p24) gag cleavage sequence renders a highly active and efficiently digested chimeric construct. The use of this insertion site, that increases the cleavage potential of this reporter enzyme, can improve the sensitivity and dynamic range of the antiviral drug assay. This simple and highly specific analytical test may also be extended to the screening of other specific protease inhibitors by a convenient colorimetric assay.
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PMID:Engineering the E. coli beta-galactosidase for the screening of antiviral protease inhibitors. 1573 8


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