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 effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.
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PMID:Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate. 170 9

Infection of embryonic bovine lung (EBL) cells by bovine immunodeficiency-like virus (BIV) were monitored by reverse transcriptase (RT), syncytia formation and polymerase chain reaction (PCR). Infection can be detected by PCR at 24 h while the presence of syncytia and RT were not detected until much later. The detection of BIV RT can be optimized by changing the pH and salt conditions. The enzyme is very sensitive to changes in pH but can tolerate a wider range of salt and MgCl2 concentrations. Infection of primary human cell cultures by BIV was monitored by both PCR and RT. No active infection of human cells were detectable.
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PMID:Comparative evaluation of bovine immunodeficiency-like virus infection by reverse transcriptase and polymerase chain reaction. 171 14

Using affinity purified human immunodeficiency virus (HIV) reverse transcriptase the reaction assay conditions were determined. The optimum incorporation of dTMP into the (rA)n(dT)10 template with HIV reverse transcriptase required 6 mM MgCl2 and 80 mM KCl. The template specificity of HIV reverse transcriptase is quite different from those of the human gamma-polymerase-associated reverse transcriptase or avian virus reverse transcriptase. The preferential inhibition of HIV reverse transcriptase as compared to human gamma-reverse transcriptase was observed with several nucleoside analog triphosphates. The Ki values for thymidine triphosphate analogs with HIV reverse transcriptase ranged from 5 to 13 nM with decreasing effectiveness for 3'-fluoro greater than 3'-amino greater than 2',3'-dideoxy greater than 3'-azido groups. This study provides information on the structure activity relationships of the triphosphate analogs inhibitory effects on HIV reverse transcriptase versus human gamma-polymerase-associated reverse transcriptase, and the possible mechanisms of action of 3' azido thymidine and the 2',3'-dideoxynucleosides, and also identifies other nucleoside analogs for possible development as inhibitors of HIV.
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PMID:Human immunodeficiency virus reverse transcriptase. General properties and its interactions with nucleoside triphosphate analogs. 243 77

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

Biochemical characteristics of the RNase H activity associated with immunoaffinity purified human immunodeficiency virus (HIV) reverse transcriptase (RT) were examined. Glycerol gradient centrifugation of HIV RT resulted in a single peak of RNase H, associated with RT activity, with an apparent molecular weight of 110,000. HIV RNase H exhibited a marked substrate preference for poly(dC).[3H]poly(rG) compared to poly(dT).[3H]poly(rA). It did not hydrolyze single-stranded RNA or the DNA component of DNA.RNA hybrids. Products of the HIV RT-associated RNase H reaction consisted primarily of monomers, dimers, and trimers with 3' OH groups. This reaction was Mg2+ dependent, with greater than 90% of maximum activity at MgCl2 concentrations between 4 and 12 mM. The optimum KCl concentration for HIV RT catalyzed polymerization with a poly(rA).(dT)10 template. The optimum pH for HIV RNase H activity was between 8.0 and 8.5, in contrast to an optimum pH of 7.5 to 8.0 for HIV RT activity. The association of RNase H activity with the p66 component of HIV RT was demonstrated by activity gel analysis. These results indicate that HIV RT has an integral RNase H activity; however, some of its properties are different from those of RNase H associated with other retroviral RT's, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization.
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PMID:Human immunodeficiency virus reverse transcriptase-associated RNase H activity. 246 65

The avian RNA tumour virus structural protein p12 was purified from avian myeloblastosis virus (AMV) by nucleic acid affinity chromatography to apparent homogeneity as judged from SDS--polyacrylamide gel electrophoresis. A filter binding assay was used for the identification of p12. High concentrations of p12 precipitated nucleic acids out of solution in the absence of MgCl2. Binding of p12 to single-stranded nucleic acids protected them from digestion with nucleases and resulted in a hyperchromic effect. These phenomena were reversible in the presence of salt. The affinity of p12 to nucleic acids was determined by competing for the binding of p12 to denatured radioactive DNA by various other nuclei acids. It was found that p12 bound preferentially to single-stranded nucleic acids and showed a higher affinity to poly(rI) than to poly(rC) and poly(rA). Purified RNA-dependent DNA polymerase activity from AMV was stimulated up to sixfold by p12, depending on the template. Solubilization of RNA in RNA--DNA hybrids by RNase H was inhibited in the presence of p12.
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PMID:Properties of the avian viral protein p12. 616 96

We have expressed and purified from Escherichia coli a human immunodeficiency virus type 1 (HIV-1) RNase H domain consisting of amino acids 400 to 560 of reverse transcriptase with either an N- or C-terminal polyhistidine tag. The native protease cleavage site of HIV-1 reverse transcriptase is between amino acids 440 and 441. Purification on Ni(2+)-nitrilotriacetate agarose resulted in a highly active RNase H domain dependent on MnCl2 rather than MgCl2. Activity was unambiguously attributed to the purified proteins by an in situ RNase H gel assay. Residues 400 to 426, which include a stretch of tryptophans, did not contribute to RNase H activity, and the polyhistidine tag was essential for activity. Despite the requirement for a histidine tag, the recombinant RNase H proteins retained characteristics of the wild-type heterodimer, as determined by examining activity in the presence of several known inhibitors of HIV-1 RNase H, including ribonucleoside vanadyl complexes, dAMP, and a monoclonal antibody. Importantly, the isolated RNase H domain produced the same specific cleavage in tRNA(3Lys) removal as HIV-1 heterodimer, leaving the 3'-rA (adenosine 5' phosphate) residue of a model tRNA attached to the adjacent U5 sequence. This HIV-1 RNase H domain sedimented as a monomer in a glycerol gradient.
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PMID:Purification and characterization of an active human immunodeficiency virus type 1 RNase H domain. 768 7

Although detection of hepatitis C virus RNA with polymerase chain reaction has become the standard for diagnosis, extensive application has been thwarted by polymerase chain reaction's labor intensiveness, risk of false-positive results through contamination and time required for individual assays. To minimize these limitations, we developed and validated a one-step hepatitis C virus RNA polymerase chain reaction assay. The one-step method was compared with traditional hepatitis C virus RNA polymerase chain reaction using primers from the highly conserved 5' untranslated region of the hepatitis C virus genome. Variables studied in the one-step method included the source and quantity of reverse transcriptase (RTase), the concentration of MgCl2 and the duration of reverse transcription and complementary DNA amplification cycles. Optimal conditions for the one-step method were obtained with 25 U of reverse transcriptase and 2 mmol/L MgCl2. The one-step method substantially reduced the time required for analysis. The sensitivity of the one-step method was comparable to that of traditional hepatitis C virus RNA polymerase chain reaction using serially diluted RNA extracted from the serum of a hepatitis C virus-infected patient. The specificity of the one-step method was confirmed on Southern-blot hybridization. The results exhibited 100% concordance with results of traditional hepatitis C virus RNA polymerase chain reaction in 50 serum samples, including those of positive and negative controls. In addition, 100% concordance was observed between the two methods' results when sera containing low levels of hepatitis C virus RNA were used.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:One-step RNA polymerase chain reaction for detection of hepatitis C virus RNA. 768 79

The interactions of HIV-1 reverse transcriptase (HIV-1 RT) with a synthetic 53/19-mer DNA substrate was investigated. For this template-primer HIV-1 RT displayed a Km value of 20 nM. The 53/19-mer competitively inhibited DNA synthesis performed on poly (rC).oligo(dG) with Ki value of 260 nM. This corresponded well to an equilibrium dissociation constant (Kd) of 300 nM, as determined by analytical ultracentrifugation. Since the Kd value is considerably higher than the corresponding Km value it is concluded that the enzyme--DNA complex is further stabilized by the binding of a cognate deoxynucleoside triphosphate and/or catalytic turnover. The association kinetics of HIV-1 RT with the 53/19-mer was measured by the fluorescence stopped-flow technique. RT bound the 53/19-mer with a rate constant of 2 +/- 1 x 10(8) M-1 s-1. The DNA binding step was succeeded by a concentration-independent step with a rate constant of 1.0 +/- 0.5 s-1 suggesting a conformational change of the enzyme. Template-primer binding of RT was influenced by the concentration of MgCl2, displaying a 17-fold increase in the Kd value when Mg2+ was increased from 1 mM to 30 mM. Since neither the association rate constant nor the conformational change was notably affected by changes of the Mg2+ concentration, it is concluded that the dissociation constant is increased by higher concentrations of Mg2+.
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PMID:Two step binding of HIV-1 reverse transcriptase to nucleic acid substrates. 769 Apr 70

A single-step reverse transcription polymerase chain reaction (SRT-PCR) method was optimized for hepatitis C virus (HCV) RNA detection. Extraction procedures by proteinase K and guanidinium isothiocyanate gave similar results. The optimal MgCl2 concentration for the SRT-PCR method was 2 mM with 10 units of superscript M-MLV RNase H-reverse transcriptase and 1 unit of Taq polymerase. Shorter PCR cycling steps gave a 10-fold-increased PCR product compared with longer cycling steps. Twenty-five anti-HCV-positive sera from chronic hepatitis C patients were positive with SRT-PCR, whereas only 17 out of 25 were positive by dissociated RT and PCR (dRT/PCR). Specificity was assessed by twenty negative controls. SRT-PCR was 5-fold more sensitive (5 HCV RNA copies per assay) than dRT/PCR with an HCV RNA transcript. Our SRT-PCR method for HCV RNA detection appears fully adapted for routine use in a medical virology laboratory.
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PMID:Detection of hepatitis C virus RNA by a reliable, optimized single-step reverse transcription polymerase chain reaction. 857 10


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