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: UNIPROT:Q07644 (polypeptide)
72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymatically active retroviral proteinases are dimers of identical polypeptide chains with a fold similar to that of other aspartic proteinases. Each polypeptide chain, encoded on one of the viral polyproteins, is less than half the size of cellular aspartic proteinases and contains only one of the two active-site aspartate residues. A plasmid was constructed to generate a genetically linked dimer of the proteinase (PR) of human immunodeficiency virus (HIV) type 1, composed of two copies of the PR sequence linked by a structurally flexible hinge region. The expression product was stable and active against HIV polyprotein substrates. Mutational analysis revealed that the linked dimer, and not multimers thereof, contained the proteolytic activity. Expression of the linked dimer as a component of a HIV polyprotein by in vitro translation gave rapid autocatalytic processing, whereas the wild-type polyprotein was stable on prolonged incubation. Transfection of HIV subviral or proviral constructs, containing the linked dimer of HIV PR, gave premature processing of the viral polyproteins, thus preventing particle formation and infectivity. Premature processing also led to increased cell toxicity.
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PMID:Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. 201 42

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.
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PMID:Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and CD4 binding. 207 45

The envelope proteins of retroviruses are derived from a polypeptide precursor protein by cleavage adjacent to a cluster of basic amino acids. Site-specific mutagenesis was used to construct a mutant of the human immunodeficiency virus type 1 (HIV-1) in which the arginine residue at the carboxy-terminus of the gp120 was changed to a threonine residue. This single substitution was sufficient to abolish all detectable cleavage of the gp160 envelope precursor polypeptide as well as virus infectivity. The gp160 was produced in normal quantities from a biologically active clone of the mutant virus after transfection into cos-1 cells. The mutant gp160 contained N-linked oligosaccharide chains with mannose-rich cores similar to those of the gp160 produced by the wild-type clone. Immunofluorescence assays showed that gp160 was transported to the surface of transfected CD4+ HeLa cells. No envelope proteins of known size could be detected in the media of cells transfected with the mutant virus, suggesting that functional virions were not formed. Binding of the mutant gp160 to the CD4 receptor molecule was unimpaired. Despite this and the presence of gp160 on the cell surface, neither growth of mutant-transfected CD4+ HeLa cells nor cocultivation of transfected cos-1 cells with H9 cells resulted in significant syncytium formation. The data indicate that the carboxy-terminal arginine residue of HIV-1 gp120 is necessary for envelope protein cleavage and suggest cleavage is important in the virus life cycle in both functional virus release and membrane fusion.
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PMID:Characterization of an HIV-1 point mutant blocked in envelope glycoprotein cleavage. 210 82

The 1.4-kb mRNA of visna lentivirus is expressed early during the lytic infection of sheep choroid plexus cell cultures. It encodes for visna early gene 1 (VEG1) product, since renamed rev gene product (or Rev), based on significant amino acid sequence homologies between this protein and the proteins of simian immunodeficiency virus of macaque and human immunodeficiency virus type 2. In this report, we examined the subcellular localization and time course appearance of the Rev protein in visna virus-infected cells. Immunoprecipitation assays of [35S]methionine-labeled cell lysates with antisera raised against the Rev protein revealed a polypeptide of 19 kDa (p19rev). This protein was predominant early in the viral replication cycle and accumulated preferentially in the cytoplasmic/membrane fraction of infected cells. Indirect immunofluorescence staining of infected cells confirmed the cytoplasmic location of visna Rev protein and could reveal in some stained cells a higher concentration of Rev at the cellular plasma membrane. The regulating protein, still present late in the viral lytic cycle, is packaged into mature viral particles along with the structural gag and env gene products.
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PMID:Subcellular localization of rev-gene product in visna virus-infected cells. 216 58

Full-length (86-residue) polypeptide corresponding to the human immunodeficiency virus type 1 tat trans-activating protein was chemically synthesized on a semiautomated apparatus, using an Fmoc amino acid continuous-flow strategy. The bulk material was relatively homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, and it showed trans-activating activity when scrape loaded into cells containing a human immunodeficiency virus long terminal repeat-chloramphenicol acetyl-transferase reporter plasmid. Reverse-phase high-pressure liquid chromatography yielded a rather broad elution profile, and assays across the column for biological activity indicated a sharper peak. Thus, high-pressure liquid chromatography provided for enrichment of biological activity. Fast atom bombardment-mass spectrometry of tryptic digests of synthetic tat identified several of the predicted tryptic peptides, consistent with accurate chemical synthesis.
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PMID:Chemical synthesis of biologically active tat trans-activating protein of human immunodeficiency virus type 1. 218 78

A simple Escherichia coli system has been developed for the detection of human immunodeficiency virus (HIV) protease activity. In this system, the protease sequence is placed downstream of the HIV gag polypeptide in an operon arrangement. Upon expression of the operon, gag serves as the substrate for the protease; the level of protease activity can be determined by measurement of the cleavage product of gag in cell extracts by Western immunoblotting. This system is useful in both detection of protease mutations generated by mutagenesis and in testing substrate specificity of the protease by mutagenesis of the gag sequence. Using this system, we have observed that modification of the N-terminus of HIV protease renders the enzyme temperature sensitive; the temperature sensitivity is made more pronounced by the conserved change of valine to isoleucine at residue eleven.
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PMID:A simple Escherichia coli system for monitoring HIV protease activity: analysis of two temperature-sensitive protease mutants. 218 4

Human hepatitis B virus (HBV) X-gene, previously shown to be capable of trans-activating heterologous regulatory elements of the human beta-interferon gene, the human immunodeficiency virus type I (HIV-1) long terminal repeat (LTR), the simian virus 40 (SV40), and HBV, has the capacity to code for a 17-kDa polypeptide (designated pX17). We now report that pX17 synthesized in Escherichia coli can activate transcription controlled by the HIV-1 LTR using a protoplast fusion technique. Protoplasts of E. coli-containing presynthesized X-protein were fused with lymphocytic H938 cells harboring an integrated copy of a plasmid with the CAT gene under control of the HIV-1 LTR (HIV-1 LTR CAT) and a marked increase in the steady state expression of the CAT mRNA was observed. When the same fused cells were treated with the protein synthesis inhibitor cyclohexamide, the pX17-dependent activation of the HIV-1 LTR was abolished. This result indicates that the X-protein expressed in E. coli is biologically active and suggests that the HBV X-protein-mediated trans-activation of the HIV-1 LTR in this system requires de novo cellular protein synthesis.
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PMID:Transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat by hepatitis B virus X-protein requires de novo protein synthesis. 219

A synthetic DNA fragment encoding a protease precursor of the human immunodeficiency virus type 2 (HIV2) was cloned and expressed in bacteria and yeast. A recombinant plasmid encoding a hybrid polypeptide consisting of human superoxide dismutase and an HIV2 protease precursor of 113 amino acids was constructed for regulated intracellular expression in bacteria. Induction of this plasmid produced an autoprocessed form of the retroviral enzyme possessing the correct molecular weight. Overexpression and secretion of the protease from yeast was achieved with an expression vector encoding the yeast pheromone alpha-factor signal/leader sequence fused to a protease precursor of 115 amino acids. Amino-terminal sequence analysis confirmed that the viral enzyme exported from yeast was correctly processed from its precursor by cleavage of the predicted Ala-Pro peptide bond located at the NH2 terminus of the protease in the pol open reading frame. No additional amino acid residues were required at the COOH terminus of the protease for this autoproteolytic event. The HIV2 protease expressed in bacteria and yeast was active in an in vitro assay when tested on the HIV1 polyprotein precursor, myristylated Pr53gag. Two synthetic peptides representing junction sequences in the HIV1 gag-pol precursor were used to assay purified HIV2 protease. The enzyme exhibited a kcat/KM of 23.2 min-1 mM-1 on the HIV1 matrix-capsid junction peptide and a kcat/KM of 71.4 min-1 mM-1 on the protease-reverse transcriptase junction peptide. These rates show that the HIV2 enzyme is efficient at hydrolyzing the HIV1 peptide junctions, revealing the analogous nature of the substrate specificities of the two enzymes.
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PMID:Recombinant HIV2 protease processes HIV1 Pr53gag and analogous junction peptides in vitro. 219 46

The predictive values of positive and negative test results for human immunodeficiency virus (HIV) antibody are extremely high in laboratories that have good quality control and high performance standards and use licensed FDA-approved enzyme immunoassay (EIA) and Western blot standardized tests. With a carefully designed protocol, the false-positive rate of combined EIA and Western blot has been reported to be as low as 1 in 10(5). When results of HIV confirmatory antibody tests are indeterminate, other tests such as culture and nucleotide probe methods for HIV DNA or RNA may help resolve false-reactive screening EIA tests. Improvements are constantly in progress for HIV laboratory tests with the use of recombinant DNA-derived antigens and synthetic polypeptides. With the use of new-generation synthetic polypeptide antigens, specific assays to identify HIV-1 and HIV-2 have been developed. Recently, assays for the HIV regulatory gene products have been studied for their predictive potential. Antibodies to nef protein, a regulator of HIV-1 replication, may be an early indicator of HIV infection.
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PMID:Current status of clinical laboratory tests for the human immunodeficiency virus. 220 98

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.
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PMID:Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease. 221 28


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