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: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for producing and purifying recombinant HIV-1 and HIV-2 reverse transcriptase (RT) is described. These enzymes are produced by Escherichia coli-transformed with a plasmid containing the gene encoding for either the human immunodeficiency virus type 1 (HIV-1) or HIV-2 RT protein. Both proteins are partially processed by host cell proteases giving rise to a mixture of heterodimeric and nonheterodimeric products, which are subsequently resolved to near homogeneity by chromatography on phosphocellulose, Q-Sepharose, and hydrophobic interaction HPLC. Both HIV-1 (66/51 kDa) and HIV-2 (68/54 kDa) heterodimeric enzymes devoid of excess unprocessed (p66 or p68) precursors are isolated, enabling comparative enzymatic characterization of the fully active (and biologically relevant) heterodimeric forms. Homogenous HIV-1 and HIV-2 RT purified by this methodology exhibit near equivalent polymerase and RNase H activities.
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PMID:Comparative purification of recombinant HIV-1 and HIV-2 reverse transcriptase: preparation of heterodimeric enzyme devoid of unprocessed gene product. 128 95

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C-terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.
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PMID:Monoclonal antibodies define linear and conformational epitopes of HIV-1 pol gene products. 171 17

To study the local immune response to human immunodeficiency virus type 1 (HIV-1) in women infected by or exposed to HIV-1, 75 women were studied: 15 were IgG-seropositive but clinically asymptomatic, 15 had acquired immune deficiency syndrome (AIDS), 15 were IgG-seronegative with seropositive husbands, and 30 were healthy seronegative women who were selected as controls. Serum samples and vaginal secretions were tested for antibodies to HIV-1 IgG and IgA by Western blot analysis. Antibodies of the IgG and IgA classes were detected in serum samples and vaginal secretions from healthy seropositive women and from women with AIDS. Local IgG antibodies to all viral proteins were detected by Western blot tests. Genital IgA antibodies were mainly directed to the core proteins p18 and p25, the p68 reverse transcriptase, and the gp160 and gp41 glycoproteins; IgA antibodies to the glycoprotein gp120 were rarely recovered. Antibodies of both the IgG and IgA classes in genital secretions were directed to all viral proteins, including surface glycoproteins, and could play a role in limiting the virus infectivity on normal mucosa.
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PMID:Antibodies to human immunodeficiency virus in vaginal secretions of heterosexual women. 276 Apr 96

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.
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PMID:Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase. 751 79

Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.
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PMID:The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases. 753 31

Serum specimens from 66 HIV-1-infected subjects were tested by ELISA for the presence of IgA antibodies to HIV-1: 44 samples were found positive and 37 were confirmed by immunoblot. In these subjects, the presence of anti-HIV IgA antibodies was studied in relation to the total count of circulating CD4+ lymphocytes and to the level of serum IgA. A significative correlation (P < 0.03) was found between the absence of IgA to the subunit p68 of the reverse transcriptase and a count of CD4+ cell < 400/mm3 or total IgA level over 4.25 g/l. The same pattern was observed for the IgA antibodies to the p52 subunit but the association was just not significant (P < 0.07). No significant decrease was noted for the IgA directed towards the other proteins of HIV-1, especially the products of the gag gene.
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PMID:Prognostic value of serum immunoglobulin A antibodies to pol gene products during HIV-1 infection. 809 40

Recently we demonstrated that the p58 subunit of p68/p58 HIV-2 reverse transcriptase (RT) heterodimer, produced by processing of p68/p68 homodimer with recombinant HIV-2 protease, terminates at Met484 [Fan, N., et al. (1995) J. Biol. Chem. 270, 13573-13579]. Here we describe purification and characterization of the p68/p58 heterodimer of recombinant HIV-2 RT. It exhibited both RT and RNase H activities, obeyed Michaelis-Menten kinetics, and was competitively inhibited by the DNA chain terminator ddTTP (Ki[app] = 305 +/- 20 nM). The HIV-2 RT-associated RNase H exhibited a marked preference for RNA hydrolysis from a HIV-1 gag-based heteropolymeric RNA/DNA hybrid in the presence of either Mg2+ or Mn2+, compared to the [3H]poly(rA).poly(dT) or [3H]poly(rG).poly(dC) homopolymeric substrates. Relative to HIV-1 RT, the RNase H activity of HIV-2 RT was only 5% toward the [3H]poly(rA).poly(dT) in the presence of Mg2+. The size distribution of products generated from [3H]poly(rA).poly(dT) by HIV-2 RT-associated RNase H was markedly distinct from that of HIV-1 RT in the presence of Mg2+ or Mn2+. The p68/p58 HIV-2 RT heterodimer, produced by specific cleavage using HIV-2 protease, should be useful for inhibition and biophysical studies aimed at discovering and designing drugs directed toward HIV-2.
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PMID:Characterization of the p68/p58 heterodimer of human immunodeficiency virus type 2 reverse transcriptase. 863 74

We isolated DEAD-box genes from three ascidian species (Ciona intestinalis, Ciona savignyi, and Halocynthia roretzi) by polymerase chain reaction methods. We obtained two types from each of C. intestinalis and C. savignyi, and four types from H. roretzi. The first type (DEAD1) belonged to the vasa subfamily, the second type (DEAD2) to the PL10 subfamily, the third type (DEAD3) to the p68 subfamily, and the forth type (DEAD4) did not belong to any of the subfamilies. We further analyzed in detail the expression pattern of C. intestinalis vasa-type gene (Ci-DEAD1) by in situ hybridization. In sections of the ovary and testis, the Ci-DEAD1-specific probe reacted intensely to small germ cells, oogonium, and/or oocyte and spermatogonium and/or spermatocyte, respectively. In whole-mount specimens of juveniles this probe specifically reacted to the primordial germ cells in the gonad rudiment. These gonad-specific expressions were confirmed by reverse transcriptase polymerase chain reaction of RNA from various tissues. The transcript was present in unfertilized eggs and in the central cytoplasm of blastomeres until the two-cell stage. During the second cleavage a part of the transcripts moved to the posterior region of embryos and, during early embryogenesis, was localized in the posterior-most blastomeres. In the tailbud, one or two hybridization signals were detected in the caudal endodermal strand. Based on these observations, we propose precursors of primordial germ cells in ascidians.
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PMID:Characterization of an ascidian DEAD-box gene, Ci-DEAD1: specific expression in the germ cells and its mRNA localization in the posterior-most blastomeres in early embryos. 1066 49

A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli. The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein. Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E. coli proteases to the heterodimer (p68/p55). This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues. The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity. The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.
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PMID:Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase. 1250 79

Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.
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PMID:Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription. 1280 62


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