Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene encoding the major envelope glycoprotein of the HIV-SF2 isolate was engineered for the secretion of recombinant gp120 (rgp120SF2) from permanent Chinese hamster ovary (CHO) cell lines. Cellular production methods were scaled up and a method for purification of the secreted glycoprotein was devised. Mild purification conditions were selected in order to preserve the native structure of the protein. rgp120SF2 exhibits a molecular weight of 120 kDa in reduced or nonreduced SDS gels; thus the polypeptide chain is intact. Deglycosylated rgp120SF2 has the predicted molecular weight of the polypeptide backbone, 54 kDa. Gel-filtration HPLC in a nondenaturing buffer at neutral pH yields a molecular weight estimate of approximately 120 kDa. Purified rgp120 closely resembles authentic viral gp120 by several physical, chemical, and immunochemical tests. rgp120SF2 reacts strongly with human HIV-positive sera, monoclonal antibodies reactive with HIV-SF2 and HIV-MN viral envelope, and a human virus-neutralizing monoclonal antibody that maps to a conserved discontinuous epitope on HIV-1 gp120. Purified rgp120SF2 forms a 1:1 molecular complex with soluble recombinant human CD4 (rCD4) receptor, as demonstrated by gel-filtration HPLC; binding is high affinity (Kd approximately 2 x 10(-9) M).
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PMID:Nonaffinity purification of recombinant gp120 for use in AIDS vaccine development. 814 40

The negative factor, Nef, of HIV-1 was found to associate to an extent of 16-42% with the detergent insoluble cytoskeletal fraction of T lymphocytes. Furthermore, Escherichia coli expressed Nef protein was found to bind during in vitro reactions with the cytoskeletal matrix to an extent of 30-50%. Cytoskeletal association of Nef was significantly enhanced by myristoylation. The specificity of the myristoylation-enhanced binding was demonstrated by the lack of an effect of myristoylation on binding of the HIV-1 Gag protein to the cytoskeleton. Cytoskeletal binding was saturable, and inhibited by high concentrations of sodium chloride, or with SDS or urea. Binding of Nef to the cytoskeletal matrix may be important in mediating its effects on HIV-1 replication.
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PMID:Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix. 821 77

Based on our findings that HIV-1 gp41 independently of CD4 can bind to the helper T cell line H9 and B cell line Raji, we characterized the putative binding of HIV-1 gp41 to the monocyte cell lines U937 and HL60. Using flow cytometry (FACS) we examined the binding of soluble gp41 (sgp41; env amino acids 539-684) to these monocyte cell lines. Using sgp41 attached to Sepharose beads, U937 cell lysates were absorbed. The sgp41 eluate of U937 cell lysates could inhibit sgp41 binding to U937 cells. With SDS-PAGE of sgp41 eluate of U937 cell lysates, three strong protein bands, (37, 45 and 62 kDa) and two weak bands (49 and 92 kDa) were stained with Coomassie blue. With Western blot (ligand blot) analysis using sgp41, three strong protein bands (37, 49 and 62 kDa) and a very weak band (42-45 kDa) were observed in sgp41 eluate of U937 cell lysates. The results suggest that the four proteins 37, 42-45, 49 and 62 kDa in U937 cell lysates are possible candidates for the putative gp41 receptor(s). We compared the blocking activities of sgp41 eluates from different cell lysates. Not only U937 and Raji lysate-sgp41 eluates, but also H9 and HL60 lysate-sgp41 eluates could block sgp41 binding to U937 and Raji cells. The results indicate that the sgp41-binding proteins on U937, or Raji (H9 and HL60, respectively) probably could have an identical blocking (or binding) specificity; these cell types carry very similar receptor(s) for HIV-1 gp41 binding.
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PMID:The human monocyte cell line U937 binds HIV-1 gp41 by proteins of 37, 45, 49, 62 and 92 kDa. 822 5

A large-scale immunoaffinity (IA) purification process was developed for the isolation of recombinant soluble antigen CD4 (sCD4) from Escherichia coli fermentations. The monoclonal antibody used for IA purification of sCD4 recognized a conformation-dependent epitope on the surface of domain 1 of CD4. IA chromatography was used to purify both sCD4-183, consisting of the N-terminal 183 amino acids of human CD4, and sCD4-PE40, a fusion protein consisting of the N-terminal 178 amino acids of CD4 and amino acids 1-3 and 253-613 of Pseudomonas exotoxin A (PE40). sCD4-183 was purified from E. coli cell pellets using cell disruption, protein solubilization, oxidation, Q-Sepharose anion-exchange and IA chromatography steps. sCD4-PE40 was purified from cell pellets using cell disruption, protein solubilization, oxidation, Cu(2+)-immobilized metal-affinity chromatography, anion-exchange and IA chromatography steps. The IA-purified sCD4 analogues demonstrated the correct apparent molecular masses on SDS/PAGE. The immobilized monoclonal antibody appeared to select for correctly folded CD4 protein, since sCD4-183 and sCD4-PE40 purified by the IA method bound human-immunodeficiency-virus glycoprotein gp120 (HIV gp120) in vitro. sCD4-PE40 purified by IA chromatography also inhibited protein synthesis in CV-1 cells expressing HIV gp120/160 at the cell surface. Relatively high recoveries of sCD4-183 and sCD4-PE40 were observed in the IA step of the purification process (71 and 79% recovery respectively). The results demonstrate that immobilized monoclonal antibodies directed against conformational epitopes may be used for rapid purification of gram amounts of correctly folded protein from mixtures of oxidized E. coli proteins.
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PMID:Large-scale immunoaffinity purification of recombinant soluble human antigen CD4 from Escherichia coli cells. 829 11

Putative cell surface human immunodeficiency virus (HIV) gp41 receptor proteins of 45 and 80 kDa (p45 and p80, respectively) were identified on human cells using a 17-amino acid peptide, referred to as CS3. In contrast, murine P815 cells expressed a peptide binding protein of 80 kDa only. A segment of 8 amino acids within CS3 contains the minimum sequence able to inhibit binding of radiolabeled CS3 to p80 and p45, as shown by competitive binding studies. Human p45 was purified from CD4+ RH9 cells by CS3 peptide affinity chromatography. Human p80 was partially purified from RH9 cell lysates by size exclusion chromatography followed by SDS-polyacrylamide gel electrophoresis; a rabbit polyclonal antibody was raised against this preparation. Anti-p80 antibody inhibited HIV infection in a dose-dependent manner. The CS3 region of gp41 has been been shown previously to be exposed on viral particles and envelope-expressing cells predominately after conformational changes in the HIV envelope occur due to the interaction of CD4 with gp120. These results, together with those from previous studies, suggest that following the interaction of gp120 with CD4, there may be a second receptor interaction necessary for virus entry/fusion.
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PMID:A peptide inhibitor of human immunodeficiency virus infection binds to novel human cell surface polypeptides. 832 99

The single-stranded nucleocapsid protein that coats the RNA genome of human immunodeficiency virus within the virion core has been produced in Escherichia coli and purified to homogeneity. The mature 55-amino acid protein, normally generated from the gag polyprotein precursor by HIV protease-catalyzed processing of both its amino and carboxyl termini, was produced in E. coli with authentic termini directly, without the need for processing. The protein was purified 30-fold to apparent homogeneity, as determined by both amino acid analysis and SDS-polyacrylamide gel electrophoresis. Sequencing of each terminus of the purified protein indicated that no proteolytic degradation occurred. A molar extinction coefficient (epsilon 280 = 8350 cm-1 M-1) was determined. The purified nucleocapsid protein binds tightly to single-stranded RNA as judged by a nitrocellulose filter binding assay. A binding constant (Kw) of 1 x 10(8) M-1 was calculated. Using fluorescence quenching of nucleocapsid protein upon RNA binding as an assay, a binding site size of seven nucleotides was determined. These results contrast to a larger 15-nucleotide site measured by others for a larger form of nucleocapsid protein-containing sequences from its immature precursor. The possible relevance of these findings are discussed.
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PMID:HIV nucleocapsid protein. Expression in Escherichia coli, purification, and characterization. 834 33

The third variable domain (V3 domain) of HIV-1 gp120 is involved in virus neutralization by antibody, in determination of cell tropism, and in syncytium-inducing/non-syncytium-inducing capacity. Antibodies are highly specific tools to delineate the role of different V3 amino acid sequences in these processes, and to dissect events occurring during synthesis of gp120/160, gp120-CD4 interaction, cellular infection, and syncytium formation. We describe here an IgG1 murine monoclonal antibody (MAb), coded IIIB-V3-01, that was raised with a synthetic peptide (FVTIGKIGNMRQAHC) derived from the carboxy-terminal flank of the HIV-1 IIIB V3 domain. The binding site of this antibody was mapped to the sequence IGKIGNMRQ, using Pepscan analysis. In ELISA, this antibody binds to E. coli-derived gp120 from HIV-1 IIIB, which is denatured and not glycosylated. The antibody showed no neutralizing activity against HIV-1 IIIB, MN, SF2, or RF in a virus neutralization assay and in a syncytium formation inhibition assay. In addition, this antibody did not react with gp120 expressed on the surface of IIIB-infected MOLT-3 cells in FACS analysis. To assess whether the epitope defined by MAb IIIB-V3-01 is hidden on native gp120, reactivity of the antibody with SDS-DTT-denatured or DTT-denatured glycosylated gp120 (CHO cell produced) was tested. Both these treatments exposed the epitope for binding. From these data we conclude that the epitope defined by MAB IIIB-V3-01 is hidden on glycosylated recombinant gp120, and is not accessible on gp120 expressed on the membrane of HIV-1, IIIB-infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A hidden region in the third variable domain of HIV-1 IIIB gp120 identified by a monoclonal antibody. 836 65

The HIV-1-specific virus protein U (Vpu) is an amphipathic membrane-associated phosphoprotein that probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. Because of an as yet undefined cytotoxic effect and the low concentration of Vpu in host cells, it has not been possible to obtain sufficient quantities of this protein for biochemical and structural investigations. We describe the synthesis, in two forms, of 50-residue peptides representing the polar cytoplasmic domain of Vpu: pVpu, comprising the wild-type Vpu sequence of HIV-1, strain HTLVIIIB, from position Ile32 to Leu81, and a mutant, pVpum2/6, with replacement of Ser52-56 with Asn. This mutation affects the two phosphorylation sites of Vpu for casein kinase-2 (CK-2), the enzyme that phosphorylates Vpu in HIV-1-infected cells. The peptides were purified to homogeneity and characterized by N-terminal sequencing, mass spectrometry and SDS-PAGE. Furthermore, both peptides were immunologically characterized by Western blot and ELISA using Vpu-specific monoclonal and polyclonal antibodies. Recombinant human CK-2 caused in vitro phosphorylation of pVpu, but had no effect on the mutant pVpum2/6. Investigation of the peptides by circular dichroism showed that addition of trifluoroethanol stabilized the alpha-helical secondary structure. Preliminary 1H NMR spectroscopic data indicated that, in the presence of trifluoroethanol, both peptides in solution have stable secondary structures with considerable alpha-helical content.
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PMID:Synthesis and characterization of the hydrophilic C-terminal domain of the human immunodeficiency virus type 1-encoded virus protein U (Vpu). 838 59

Based on our findings that HIV-1 gp41 independently of CD4, can bind to the helper T-cell line H9, we characterized putative binding of HIV-1 gp41 to B-cell lines, Raji, Bjab and Ramos. Using fluorescence-activated cell sorter (FACS) we examined the binding of soluble gp41 (sgp41; Env amino acid 539-684) to these B-cell lines. Using sgp41 attached to sepharose beads Raji cell lysates were absorbed. The sgp41-eluate of Raji cell lysates could inhibit the sgp41-binding to Raji cells. By SDS-PAGE of sgp41-eluate of Raji cell lysates four strong protein band, 37, 45, 49 and 62 kD, and a weak band of 92 kD were stained with Coomassie blue. By Western blot (ligand blot) analysis using sgp41 four protein bands, 37, 45, 49 and 62 kD, were observed in sgp41-eluate of Raji cell lysates. To test the individual proteins the five proteins were isolated from the sgp41-eluate of Raji cell lysates. Three proteins, 45, 49 and 62 kD, each could partially inhibit the sgp41-binding to Raji cells. The results suggest that these three proteins in Raji cell lysates are possible candidates for the putative gp41 receptor(s).
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PMID:HIV-1 gp41 binds to several proteins on the human B-cell line, Raji. 841 20

The imino sugar N-butyldeoxynojirimycin (NB-DNJ) exhibits anti-HIV activity in vitro and inhibits the purified glycoprocessing enzyme alpha 1,2-glucosidase I. It has been speculated that the anti-viral activity of this compound may result from inhibition of HIV envelope glycoprotein processing. However, structural evidence that glucosidase inhibition takes place in intact cells at the anti-viral concentration (0.5 mM) is lacking. In this study, N-linked glycosylation of recombinant gp120 expressed in Chinese hamster ovary cells cultured in the presence or absence of NB-DNJ has been characterized. Immunoprecipitation, in conjunction with endoglycosidase H (endo H) digestion and SDS-polyacrylamide gel electrophoresis analysis, revealed that the glycosylation of gp120 was profoundly altered in the presence of NB-DNJ. The majority of the gp120 oligosaccharides from untreated cells were resistant to endo H. However, nearly complete endo H sensitivity was observed following treatment with 0.5 mM NB-DNJ indicating that gp120 expressed in treated cells carries immature, high mannose type oligosaccharides. In addition, using metabolic labeling with [3H]mannose, gel filtration chromatography, and digestion with highly purified glucosidases I and II, we provide the first definitive evidence that glucosidase I inhibition occurs at the anti-viral concentration of NB-DNJ. These data indicate that glucosidase inhibition is a candidate mechanism for the anti-viral activity of this compound.
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PMID:Effects of the imino sugar N-butyldeoxynojirimycin on the N-glycosylation of recombinant gp120. 841 62


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