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)

The active site of human immunodeficiency virus reverse transcriptase (HIV1-RT) was probed using three group-specific reagents: phenylglyoxal (PG), N-ethylmaleimide (NEM), and pyridoxal 5'-phosphate (PLP). The inactivation of HIV1-RT by arginine-specific PG was found to be completely protected against by adding primer-template. The potential active site arginine was localized to position 277 in the primary structure, suggesting that the polymerase domain of the enzyme should be considered as extending at least this far from the N terminus. The sulfhydryl-modifying reagent NEM completely inhibits NY5-HIV1-RT, which contains a cysteine at position 162, and such inhibition is protected against by primer-template. However, it does not strongly inhibit LAV-HIV1-RT, in which C162 is replaced by S162, indicating that while C162 may be at or near the active site or interact allosterically with primer-template, it is not essential for activity. The lysine-specific reagent PLP was found to be a noncompetitive inhibitor with respect to both primer-template [poly(rA).oligo(dT)] and dTTP. The latter result differentiates HIV1-RT from other RTs, for which PLP has been shown to be a competitive inhibitor with respect to dTTP.
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PMID:Active site studies of human immunodeficiency virus reverse transcriptase. 138 Aug 26

A nonradioactive micro-assay procedure for detection of released reverse transcriptase activity from cells infected with equine infectious anemia virus is described. This procedure utilizes biotinylated-dUTP in conjunction with a streptavidin-alkaline phosphatase conjugate. Detection of alkaline phosphatase is by autoradiography of the chemiluminescence produced during enzymatic dephosphorylation of Lumi Phos 530. This method, as with reverse transcriptase micro-assays employing 32P-labeled nucleotides, is suited to the processing of numerous samples, while having the advantages of safety and stability normally associated with nonradioactive methods of detection. Sensitivity is comparable to a reverse transcriptase micro-assay using 32P-dTTP.
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PMID:A nonradioactive micro-assay for released reverse transcriptase activity of a lentivirus. 138 69

Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.
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PMID:In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome. 138 71

We have isolated a lymphoid cell line, MDS, from the pleural exudate of a patient with chronic myelomonocytic leukemia. The cells are biphenotypic, containing various T-cell and myeloid markers, and are surface negative for CD4 and CD8 but have low CD4 mRNA. The cells grow in suspension with a doubling time of 15 hr, have been karyotyped as trisomy 21, are negative for human immunodeficiency virus type 1 (HIV-1), and are tumorigenic in the nude mouse. We have isolated two stable HIV-1-producing cell lines, MDS-T, by transfecting MDS cells with pHXBc2, and MDS-I, by infecting MDS cells with HIV-1IIIB. In 24 hr, 1 x 10(5) MDS-T or MDS-I cells produce 46 ng of p24 per ml and reverse transcriptase that is capable of incorporating 0.2 pmol of [32P]TTP into oligo(dT).poly(A). Ultrastructural studies showed numerous mature viral particles in MDS-T and MDS-I cells that are capable of infecting T cells. HIV-1 infection could be inhibited by 25% in the MDS cells with the anti-CD4 antibody Leu 3a. For over a year MDS-T and MDS-I cells have been producing high concentrations of HIV-1 in culture. A subclone derived from the MDS cells behaves like the parent cells when transfected or infected with HIV-1. In contrast to other T-cell lines, neither phorbol 12-myristate 13-acetate nor tumor necrosis factor alpha stimulated the replication of HIV-1, whereas bromoadenosine 3',5'-cyclic monophosphate or interferon alpha caused 50% and 80% inhibition of reverse transcriptase production, respectively. These chronically infected T-cell lines are a useful model system to study the effect of anti-HIV agents and cellular factors required for HIV-1 replication.
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PMID:Productive nonlytic human immunodeficiency virus type 1 replication in a newly established human leukemia cell line. 143 50

2',3'-Dideoxyuridine (ddUrd) exhibits poor if any anti-human immunodeficiency virus (HIV) activity in ATH8 and MT-4 cells. This is in agreement with the failure of ddUrd to be efficiently anabolized intracellularly to its 5'-triphosphate metabolite. However, 2',3'-dideoxyuridine-5'-triphosphate (ddUTP) proved to be a potent and selective inhibitor of the reverse transcriptase of HIV (Ki, 0.05 microM) and avian myeloblastosis virus (Ki, 1.0 microM). Bacterial DNA polymerase I, mammalian DNA polymerase alpha, terminal deoxyribonucleotidyl transferase, and Moloney murine leukemia virus reverse transcriptase were resistant to ddUTP. ddUTP is incorporated into the growing DNA chain principally at dTTP sites and inhibits further elongation. The potential of ddUTP as an anti-HIV therapeutic agent merits further investigation. However, to achieve this goal, it will be necessary to resort to techniques capable of delivering preformed phosphorylated ddUrd to the susceptible cells.
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PMID:Potent DNA chain termination activity and selective inhibition of human immunodeficiency virus reverse transcriptase by 2',3'-dideoxyuridine-5'-triphosphate. 168 52

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
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PMID:Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses. 169 11

Wheat DNA polymerase A has been purified from wheat germ. The previous purification procedure (Castroviejo, M. et al. (1979) Biochem. J. 181, 183-191; Tarrago-Litvak, L. et al. (1975) FEBS Lett. 59, 125-130), has been improved leading to a higher degree of purity. Several biochemical properties of the enzyme are described. Interestingly, wheat DNA polymerase A is able to copy natural poly(A)+ mRNA into cDNA, in a way that is similar to that of the human immunodeficiency virus reverse transcriptase (HIV-RT). All four dXTP and the oligo dT primer were required for cDNA synthesis. The cDNA product was completely digested in the presence of DNase I and predigestion of the mRNA template with RNase decreased dramatically the cDNA synthesis. The animal DNA polymerase gamma can not copy natural mRNA. Substances, known to alter the enzymatic activities have been used to compare enzymes properties. In the presence of glycerol, ethidium bromide or spermine, wheat DNA polymerase A, HIV-RT and DNA polymerase gamma behave similar and they differ from animal DNA polymerase alpha. Nevertheless, DNA polymerase A is more resistant than HIV-RT and DNA polymerase gamma to the chain terminator ddTTP, while the wheat enzyme is more inhibited than DNA polymerase gamma but more resistant than HIV-RT in the presence of N3-TTP.
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PMID:Wheat embryo DNA polymerase A reverse transcribes natural and synthetic RNA templates. Biochemical characterization and comparison with animal DNA polymerase gamma and retroviral reverse transcriptase. 169 Oct 20

We engineered a prokaryotic expression vector encoding the HIV reverse transcriptase (RT). We grew Escherichia coli JM109 carrying the vector in a 250-liter stirred tank fermentor and purified RT (p66) under native conditions to apparent homogeneity. Purified p66 (greater than or equal to 5 mg/ml) was not stable, and was rapidly processed to its 51 kD derivative (p51), until p66:p51 levels were approximately 1:1. These latter RT preparations were chromatographed as heterodimers and had approximately fivefold higher specific RT enzymatic activities compared with those containing predominantly p66. P66 purified under dilute concentrations (less than or equal to 0.5 mg/ml) was monomeric in solution, resistant to p51 processing for weeks at 4 degrees C, but also had low specific RT enzymatic activities. To attempt the preparation of homogeneous p66 with specific RT enzymatic activities equivalent to p66:p51 heterodimers, purified heterodimers were denatured and p66 was purified and refolded during extensive dialysis (refolded p66). Refolded p66 (less than or equal to 0.5 mg/ml) was monomeric in solution and had identical specific RT enzymatic activities, Km for dTTP, and inhibition by 3'-azido-3'-deoxythymidine triphosphate compared with heterodimeric p66:p51 RT. The data indicates that HIV RT obtained from recombinant E. coli under native conditions is extensively processed at concentrations promoting dimerization. Moreover, RT denaturation and refolding yields apparently homogeneous monomeric p66, with specific RT enzymatic activities equivalent to heterodimeric RT.
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PMID:Denaturation/refolding of purified recombinant HIV reverse transcriptase yields monomeric enzyme with high enzymatic activity. 169 23

Three analogs of thymidine, D4T [2',3'-didehydro-2',3'-dideoxythymidine; 1-(2,3-dideoxy-beta-D-glyceropent-2-enofuranosyl)thymine], FddT (3'-fluoro-3'-deoxythymidine), and AZT (3'-azido-3'-deoxythymidine), were compared in biological tests designed to assess their potential utility as anti-human immunodeficiency virus (HIV) agents. The in vitro potencies of these compounds against HIV infection in CEM cells were measured, with FddT and AZT being more potent than D4T. The cytotoxicities of D4T, FddT, and AZT for CEM cells were comparable. The triphosphates of these three derivatives inhibited purified HIV reverse transcriptase, and their affinities for this polymerase were found to be 1 or 2 orders of magnitude greater than that for the normal substrate, dTTP. D4T was less toxic than FddT or AZT for cultured human and mouse bone marrow cells (granulocyte-macrophage CFU). The three compounds had similar toxicities for human progenitor erythrocyte burst-forming units. In a 30-day mouse toxicity study, AZT and FddT produced a similar spectrum of hematopoietic toxicities. These toxic effects occurred at much lower doses of FddT than of AZT. At the higher doses of FddT, a significant incidence of lethality occurred. By contrast, D4T was considerably less toxic than both AZT and FddT in this study. The dose-limiting toxicity of D4T in mice was hepatotoxicity. The very different phosphorylation patterns of D4T, its lower toxicity, and its comparable potency relative to FddT and AZT suggest that the potential of D4T as an anti-HIV agent should be further explored.
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PMID:Comparison of in vitro biological properties and mouse toxicities of three thymidine analogs active against human immunodeficiency virus. 169 57

The action of 3'-azido-3'-deoxythymidine 5'-triphosphate (N3dTTP) on DNA strand elongation catalyzed by human immunodeficiency virus type 1 reverse transcriptase was evaluated in comparison with human DNA polymerase alpha and proliferating cell nuclear antigen-independent DNA polymerase delta. Sequencing gel analysis demonstrated that the human immunodeficiency virus 1 reverse transcriptase preferentially incorporated N3dTTP into the T sites of the growing DNA strands and caused chain termination in a dose-dependent manner. This effect was observed even when the N3dTTP concentration was 0.3 microM, 100-fold less than dTTP. Studies with reverse transcriptases from avian myeloblastosis virus and Moloney murine leukemia virus showed that N3dTTP was also efficiently incorporated into DNA by these enzymes and terminated DNA strand elongation. In contrast, human DNA polymerases alpha and delta did not incorporate detectable amounts of N3dTTP into the DNA and were not inhibited by 300 microM N3dTTP. The selective incorporation of the chain-terminating nucleotide by the viral reverse transcriptases appears to be a molecular basis for the positive therapeutic index of 3'-azido-3'-deoxythymidine.
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PMID:Selective action of 3'-azido-3'-deoxythymidine 5'-triphosphate on viral reverse transcriptases and human DNA polymerases. 169 49


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