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Query: UMLS:C0272170 (SDS)
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The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.
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PMID:Partial purification and characterization of the reverse transcriptase of the simian immunodeficiency virus TYO-7 isolated from an African green monkey. 169 22

The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.
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PMID:The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity. 171 Dec 22

With the aid of monoclonal antibodies to the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1), low-molecular-mass subunits (p29, p32, and p40) were identified in HIV-1 RT purified from HIV (HTLV-IIIB) virions by isoelectric focusing. Epitope mapping with synthetic polypeptides from various regions of the pol gene suggests that the low-molecular-mass subunits result from N-terminal cleavage of the p51 subunit. The subunits could be separated only by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting. They could not be separated on chromatographic columns, suggesting that the subunits are complexed or conformationally arranged in such a way that their separation on the basis of molecular mass is not possible. The molecular mass of the active enzyme eluted from a chromatographic column (Sephacryl S-300) loaded with a mixture of the subunits was estimated to be 100 kDa.
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PMID:Epitope mapping of the low-molecular-mass subunits of reverse transcriptase in human immunodeficiency virus type 1 by monoclonal antibodies. 172 5

Storage of maternal mRNAs as nontranslated ribonucleoprotein (RNP) complexes is an adaptive strategy in various vertebrate and invertebrate oocytes, for rapid translational recruitment during embryonic development. Previously, we showed that Xenopus laevis oocytes have a soluble cytoplasmic pool of mRNA-binding proteins and particles competent for messenger RNP assembly in vitro. Here we report the isolation of cDNAs for the most abundant messenger RNPs, the 54- and 56-kDa polypeptide (p54/p56) components of the approximately 6S mRNA-binding particle, from an ovarian expression library. The nucleotide sequence of p56 cDNA is almost identical to that recently reported for the putative Xenopus transcription factor FRG Y2. p54 and p56 are highly homologous and are smaller than expected by SDS/PAGE (36 kDa and 37 kDa) due to anomalous electrophoretic mobility. They lack the "RNP consensus motif" but contain four arginine-rich "basic/aromatic islands" that are similar to the RNA-binding domain of bacteriophage mRNA antiterminator proteins and of tat protein of human immunodeficiency virus. The basic/aromatic regions and a second conspicuous 100-amino acid "domain C" of p54 and p56 are conserved in the following DNA-binding proteins: human proteins dpbA, dpbB, and YB-1, rat protein EFIA, and Xenopus protein FRG Y1, all reported to bind to DNA; domain C is homologous to the major Escherichia coli cold-stress-response protein reportedly involved in translational control. Antibodies raised against a peptide of domain C have identified similar proteins in Xenopus somatic cells and in some mammalian cells and tissues. We conclude that p54 and p56 define a family of RNA-binding proteins, at least some of which may be involved in translational regulation.
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PMID:Sequence analysis of cytoplasmic mRNA-binding proteins of Xenopus oocytes identifies a family of RNA-binding proteins. 172 76

The envelope (env) glycoprotein of human immunodeficiency virus 1 (HIV-1), initially synthesized as a precursor molecule termed gp160, is cleaved into two noncovalently associated subunits prior to delivery to the plasma membrane. We have studied the oligomeric structure of this protein using chemical cross-linking, velocity gradient sedimentation, and SDS-resistance. We find that gp160 forms stable homodimers after synthesis. After cleavage to gp120/gp41 the molecule becomes less stable to detergent solubilization and centrifugation but remains dimeric. Interactions between the 129 amino terminal residues in the ectodomains of adjoining gp41 subunits are both sufficient and necessary for assembly. In addition, tetramers composed of two dimers were also formed. Larger structures were not observed. The tetrameric paramyxovirus F protein, which has structural and functional similarities to the HIV-1 env protein, also forms a dimer of dimers.
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PMID:The assembly of the HIV-1 env glycoprotein into dimers and tetramers. 178 45

A total of 81 cell clones persistently infected with the LAV-1 or HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) was isolated from cells which were obtained by serial passage of some proliferating MT-4 cells after a drastic cytolysis of most cells by HIV-1-infection. These cell clones were classified into 8 types (I to VIII) in terms of the expression of HIV-1 antigens, syncytium formation capacity, and reverse transcriptase activity and infectivity of virus particles in the culture fluid. Type I cell clones were producers of infectious HIV-1 particles, while types II to VIII cell clones did not produce infectious HIV-1 or were producers of uninfectious defective HIV-1 particles. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (PAGE) showed that the gag precursor protein in L-2 cell clone (type IV) was not cleaved to mature gag proteins, while the env precursor protein on L-3 cell clone (type III) was not cleaved to mature env protein. H-7 cell clone (type VIII) did not express any HIV-1 antigen. All these cell clones after the superinfection with infectious HIV-1 synthesized intact gag and env proteins, which were, at least in part, related to the HIV-1 genome persistently present in the cell clones before the superinfection, resulting in production of infectious HIV-1. For example, it was found that L-2 cell clone contained a single copy of the LAV-1 genome per haploid cell and produced doughnut-shaped particles. On the other hand, the cell clone isolated from the L-2 cell clone superinfected with infectious HTLV-IIIB contained the integrated HTLV-IIIB genome in addition to the LAV-1 genome present before the superinfection, and produced intact HIV-1 particles in addition to doughnut-shaped particles from a single cell. These results indicate that complementation and/or genetic recombination events in the superinfected cells may account for the production of infectious intact HIV-1 virions.
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PMID:Production of infectious particles from defective human immunodeficiency virus type 1 (HIV-1)-producing cell clones by superinfection with infectious HIV-1. 200 Nov 75

Site-directed mutagenesis was used to study the biological significance of a disulphide bridge and two N-linked oligosaccharides in the CD4-binding region of the envelope glycoproteins of human immunodeficiency virus type 1. Mutagenesis was performed in a phage M13 system at sites corresponding to the cysteine residue (amino acid 402) and the asparagine residues (390 and 447) of the env gene. The mutated env gene was inserted into a recombinant vaccinia virus under the control of the vaccinia virus 7.5K promoter and the expression of mutated env proteins was analysed by SDS-PAGE, a conventional indirect immunofluorescence assay and by a fluorescence-activated cell sorter. Cysteine 402 was found to be essential for the specific cleavage of gp160 into gp120 and gp41, and for intracellular transport of the protein to the cell surface. CD4-binding and syncytium formation assays demonstrated that the disulphide bridge of cysteine 402 stabilized a conformation essential for receptor binding as well as syncytium formation by CD4+ cells. No altered biological activity compared to that of the wild-type proteins could be detected for the mutant proteins lacking the N-glycosylation sites. These data show that the two conserved glycans attached to asparagine residues 390 and 447 do not play any active role in the formation of the disulphide bridge involving cysteine 402 or in the maintenance of an active conformation of the protein, despite their location within the functionally important CD4-binding region.
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PMID:Effects of mutations in glycosylation sites and disulphide bonds on processing, CD4-binding and fusion activity of human immunodeficiency virus envelope glycoproteins. 204 92

The 95 amino acid-protein encoded by the non-structural vpr gene of the human immunodeficiency virus type 1 (LAV-1BRU isolate) was chemically synthesized by solid phase methodology. The synthetic vpr protein was characterized by amino acid analysis, sequence analysis, RP-HPLC, and urea-SDS PAGE. Using a radioimmunoassay, antibodies to the synthetic protein were detected in sera of 25% of HIV 1-seropositive patients tested. Western blot analysis suggested that the antibodies preferentially recognize the dimeric form of vpr.
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PMID:A synthetic protein corresponding to the entire vpr gene product from the human immunodeficiency virus HIV-1 is recognized by antibodies from HIV-infected patients. 214 26

We describe here a novel immunoassay procedure, designated strip-ELISA (S-ELISA), in which specific antigens are purified by SDS-PAGE, transferred to support membranes, and utilized in situ as substrate in routine ELISA procedures. Using two different lentivirus systems, simian immunodeficiency virus and equine infectious anemia virus, we demonstrate the utility of S-ELISA for screening hybridoma supernatants during production of monoclonal antibodies and for the dissection of polyclonal antibody responses into defined antigen specificities. The data presented here indicates that the S-ELISA combines the sensitivity of conventional ELISA with the specificity afforded by Western blotting procedures, producing a relatively simple and versatile assay for evaluating antibody reactivities.
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PMID:Analysis of antibody reactivities in ELISA using protein blots as antigen substrates: S-ELISA. 215 66

1) The aspartic proteinase of the human immunodeficiency virus type 1 (HIV-1) was purified from cultures of recombinant E. coli. The enzyme preparation is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing. 2) A rapid assay procedure for the proteinase was established which makes use of the cleavage of a radiolabeled decapeptide and the separation of substrate and labeled product by ion-exchange resin. 3) Activity of the enzyme is optimal at an ionic strength of 2.5-3.5M; also, the inhibitor pepstatin is a more potent inhibitor at higher ionic strength. This can be attributed to a tighter binding of both substrate and inhibitor in high-salt buffer. 4) The Km value of the decapeptide substrate is independent of the pH in the range of 3.5-7.5, while kcat shows a bell-shaped curve with a maximum at pH 5.2. The shape of the curve can be attributed to pKa values of 4.2 and 6.2 of groups on the enzyme. Pepstatin inhibition is optimal below pH 5.5, but becomes weak above pH 6.
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PMID:Purification, assay and kinetic features of HIV-1 proteinase. 218 85


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