SEARCH · PubMed Health

Results for “reverse transcriptases”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12Linked to original sources

PubMed1990

Template switching by reverse transcriptase during DNA synthesis.

The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction. In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes. The assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added. We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase. The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching. In the absence of RNase H, switching still occurred but the efficiency was lowered. Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H. Finally, we examined the action of the RNase H on RNA templates after primary transcription but prior to template switching. We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product. This result was consistent with the work of others who have shown that RNase H acts as an endonuclease.

Chromosome Deletion↗

PubMed2000

Immunomodulating effect of 2,3,4,5-tetrahydro-1H-3-benzazepines (a new class of non-nucleoside inhibitors of reverse transcriptase).

The effects of newly synthesized, reverse transcriptase inhibitors, 3-benzazepines, for their effects on natural killer (NK) cell and blast transformation in human peripheral blood mononuclear cells were investigated. The most effective reverse transcriptase inhibitors were KF1, KF2 and KF3, which primarily suppressed immunological functions. Besides the inhibition of T cell proliferation, the benzazepines also show inhibitory effect on NK cell functions, in particularly, against large granular lymphocytes and monocytes. The B lymphocytes and Fc mediated killer functions were less inhibited.

Adjuvants, Immunologic↗

PubMed1982

Differential inhibition of DNA polymerase and RNase H activities of the reverse transcriptase by phosphonoformate.

Three potential inhibitors of reverse transcriptase activities, phosphonoformate (PF), phosphonoacetate (PAA), and ethyl-diethyl phosphonoformate (Et-PF), were compared in this study. Only PF was found to inhibit the DNA polymerase activity of the purified reverse transcriptase of Moloney murine leukemia virus (M-MuLV) and avian myeloblastosis virus (AMV). The degree of DNA polymerase inhibition was linear with PF concentration; 50% inhibition was achieved at 10 muM. Whereas PF inhibited both the RNA and DNA dependent DNA polymerase activities, the RNase H activity of the reverse transcriptase was unaffected. Both the endogenous DNA polymerase activity in detergent disrupted virus and the activity of the purified enzyme with the isolated virus genome 70S RNA were inhibited by PF. However, higher concentrations of PF were needed to inhibit the endogenous reaction. The inhibition by PF appeared to be reversible and noncompetitive with respect to the substrate deoxythymidine triphosphate (dTTP). Addition of PF after the initiation of DNA synthesis immediately arrested the reaction.

Animals↗

PubMed1990

Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases.

Four flavonoids, 5,6,7-trihydroxyflavone (baicalein), 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,3',4',5,6,7-hexahydroxyflavone (quercetagetin) and 3,3',4',5,5',7-hexahydroxyflavone (myricetin), were found to be potent inhibitors of reverse transcriptases from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV). Under the reaction conditions employed, any one of these flavonoids almost completely inhibited the activity of RLV reverse transcriptase at a concentration of 1 microgram/ml. HIV reverse transcriptase was inhibited by 100%, 100%, 90% and 70% in the presence of 2 micrograms/ml quercetin, myricetin, quercetagetin and baicalein, respectively. The mode of inhibition of these flavonoids was competitive (RLV reverse transcriptase) or partially competitive (HIV reverse transcriptase) with respect to the template.primer complex, (rA)n.(dT), and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values for RLV reverse transcriptase were found to be 0.37 microM and 0.08 microM for baicalein and quercetin, respectively and those for HIV reverse transcriptase were 2.52 microM, 0.52 microM, 0.46 microM and 0.08 microM for baicalein, quercetin, quercetagetin and myricetin, respectively. Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones and flavanones) carried out to clarify the structure/activity relationships, revealed that the presence of both the unsaturated double bond between positions 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, (i.e. baicalein) were a prerequisite for the inhibition of reverse transcriptase activity. Removal of the 6-hydroxyl group of baicalein required the introduction of three additional hydroxyl groups at positions 3, 3' and 4' (quercetin), to afford a compound still capable of inhibiting the reverse transcriptase activity. Quercetagetin which contains the structures of both baicalein and quercetin, and myricetin which has the structure of quercetin with an additional hydroxyl group on the 5' position also proved strong inhibitors of reverse transcriptase activity. The inhibition by baicalein of reverse transcriptase is highly specific, whereas quercetin and quercetagetin were also strong inhibitors of DNA polymerase beta and DNA polymerase I, respectively. Myricetin was also a potent inhibitor of both DNA polymerase alpha and DNA polymerase I.

Animals↗

PubMed1975

Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase).

The ability of tryptophan tRNA (tRNATrp) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNA-dependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrp and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNAs, only tRNATrp and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATrp to change the sedimentation rate of the enzyme indicates that tRNATrp is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrp aa a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme.

Animals↗

PubMed1987

Inhibition of viral reverse transcriptase by 2',5'-oligoadenylates.

Viral reverse transcriptase activity was inhibited in a concentration dependent manner by 2',5'-oligoadenylate. Kinetically this inhibition was of a mixed type where 2',5'-oligoadenylate was not strictly competitive with dTTP. The potency of inhibition was more marked in the absence than in the presence of sulfhydryl agents. 2',5'-oligoadenylate had no effect on DNA-dependent E. coli DNA polymerase and was much less active against mammalian DNA polymerases. This is the first report of reverse transcriptase inhibition by an inducible constitutive natural ligand.

Adenine Nucleotides↗

PubMed2001

Substitution of Asp114 or Arg116 in the fingers domain of moloney murine leukemia virus reverse transcriptase affects interactions with the template-primer resulting in decreased processivity.

Reverse transcriptase, an essential retroviral DNA polymerase, replicates the single-stranded RNA genome of the retrovirus, producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. Substitution of Ala for either Asp114 or Arg116, two highly conserved residues in the fingers domain of Moloney murine leukemia virus reverse transcriptase, results in enzymes (D114A or R116A) with significant defects in their abilities to processively synthesize DNA using RNA or DNA as a template. D114A and R116A enzymes also bind more weakly to template-primer in the presence of added deoxyribonucleotides, as seen by gel-shift analysis, but retain the ability to strand transfer and accumulate smaller RNase H cleavage products when compared to the wild-type enzyme. In addition, mutant proviruses, including D114A and R116A substitutions in Moloney murine leukemia virus reverse transcriptase, are not viable despite the presence of processed reverse transcriptase in the viral particles. A potential mechanistic role in processive synthesis for D114 and R116 is discussed in the context of our results, related studies on HIV-1 reverse transcriptase, and previous structural studies.

Amino Acid Substitution↗

PubMed1988

Enzymatically active forms of reverse transcriptase of the human immunodeficiency virus.

The reverse transcriptase of HIV-1 (AIDS virus) is characterized by the presence of two highly immunogenic proteins of 66 and 51 kD known to be enzymatically active as a complex p66/51. Using an activity gel procedure that allows identification of catalytic polypeptides in situ after PAGE in denaturing conditions, we visualized two major active bands of 66 and 51 kD of reverse transcriptase from highly purified preparations of HIV-1. We show that both p66 and p51 are enzymatically active. An additional active band was also associated with a 165 kD polypeptide, representing about 2-4% of total activity and possibly corresponding to the putative gag-pol precursor. In H9-infected cells the 66 kD active band became visible 70 hours after infection. These studies show that the two major forms of reverse transcriptase (66 and 51 kD) of HIV-1 are independently active and that a higher Mr form of 165 kD is also enzymatically active.

Cell Line↗

PubMedDate not supplied

Clinical uses of non-nucleoside reverse transcriptase inhibitors.

Three non-nucleoside reverse transcriptase inhibitors (NNRTIs) are currently available for treatment of HIV-1 as part of combination antiretroviral therapy. Oral dosing is administered three times daily for delavirdine (DLV), twice daily for nevirapine (NVP), and once daily for efavirenz (EFV). Rash is a common side effect of all three NNRTIs, and early CNS side effects are also frequent with EFV. Hepatotoxicity is relatively uncommon but requires appropriate monitoring. Drug interactions mediated by the cytochrome P450 system are an important consideration when the NNRTIs are administered concomitantly with other drugs, including protease inhibitors (PIs). HIV strains with reduced susceptibility to NNRTIs can occur with a single mutation in the reverse transcriptase (RT) gene. The available NNRTIs exhibit overlapping genotypic resistance patterns, but newer agents may overcome this problem. NNRTIs have been studied in combination with nucleoside RT inhibitors for first-line HIV therapy, where they have shown at least equivalent antiviral efficacy compared with PI-based regimens over 1-2 years of therapy. NVP and EFV have also been studied as a replacement for a PI within a virologically successful regimen, with the aim of preventing or reducing PI toxicities and simplifying the dosing regimen. Such 'switch' strategies are successful for certain patients in maintaining virologic suppression for 6 months or more and result in varying degrees of improvement in PI-associated toxicities. NNRTIs may offer a benefit when included in salvage regimens for patients failing PI-based therapy, particularly in patients who have not previously been treated with NNRTIs. NVP has been shown to have a substantial favourable impact on the rate of vertical HIV-1 transmission with a simple, cost-effective regimen.

Alkynes↗

PubMed1991

Thermodynamics of A:G mismatch poly(dG) synthesis by human immunodeficiency virus 1 reverse transcriptase.

Human immunodeficiency virus 1 (HIV-1) reverse transcriptase has been found to conduct error-prone synthesis on DNA and RNA templates. We find here that tolerance of an A:G mispair with poly(rA) as template is particularly strong, such that extensive poly(dG) synthesis is conducted. This type of extensive misincorporation is not observed with several reference DNA polymerases. Surprisingly, HIV reverse transcriptase processivity and kcat for dGMP misincorporation and normal dTMP incorporation are about the same. However, the Km value for dGTP in poly(dG) synthesis is approximately 1000-fold higher than the Km for dTTP in poly(dT) synthesis. Comparison of thermodynamic parameters for dGMP misincorporation and normal dNMP incorporation indicates a lower energy of activation for dGMP misincorporation than for normal dNMP incorporation. Entropy of activation (delta S*) for normal dTMP incorporation is positive (approximately 10 cal/kmol), whereas delta S* for dGMP misincorporation is negative (-36 cal/kmol). Since differences in delta S* are usually considered to reflect differences in solvation for the transition state complex, these results are consistent with the interpretation that the active site of HIV reverse transcriptase is flexible enough to misincorporate dGMP without the usual dispersion of water molecules.

Autoradiography↗

PubMedDate not supplied

[Reverse transcriptase of the human immunodeficiency virus: isolation and substrate specificity].

Human immunodeficiency virus (HIV-I) reverse transcriptase was expressed in E. coli and purified to homogeneity (E. coli strain RRI (pRC-RT, pRK 248cIts)). We have investigated the substrate properties toward to DNA synthesis, catalyzed by this enzyme, of some nucleoside-5'-triphosphate analogues, previously studied in the same reactions, catalyzed by AMV and M-MLV reverse transcriptases. We have investigated substrate properties of new analogues of 2',3'-dideoxy-2',3'-didehydro- and 2',3'-dideoxytubercidin-5'-triphosphates. We have compared the relative efficiency of incorporation of different analogues tested in the DNA chain. It has been shown that expressed and purified HIV reverse transcriptase had the same specificity to analogues of 2'-deoxyribonucleoside-5'-triphosphates as was described for reverse transcriptases and natural HIV reverse transcriptase as well. These properties allow to apply the expressed HIV reverse transcriptase in different model systems.

Base Sequence↗

PubMed1989

[Comparative evaluation of markers with reverse transcriptase inhibiting antibody in human immunodeficiency virus type 1 infection].

To investigate a non-RI test which is equivalent to the reverse transcriptase inhibiting antibody test, a reverse transcriptase inhibiting antibody was compared to the absolute number of CD-4 or CD-8 cells or CD-4/-8 ratio and also to photodensitometric analysis for western blotting. There is no correlation of the reverse transcriptase with any test for cell numbers and their ratio. In photodensitometry, relative units of anti-p65 and anti-p51 were compared with reverse transcriptase inhibiting antibody. The reverse transcriptase inhibiting antibody showed a higher correlation to the relative unit of p65 antibodies than that of p51 antibodies. The photodensitometric analysis of western blotting for a serum test may be a possible method to find a prognostic marker in HIV-1 infection.

Acquired Immunodeficiency Syndrome↗

PubMed1980

Simple affinity procedure for the purification of mammalian viral reverse transcriptases.

Polyguanylic acid was found to be a potent inhibitor of RNase H associated with mammalian viral reverse transcriptase, indicating a strong interaction between polyguanylic acid and the reverse transcriptase protein. Based on this observation, we have developed three simple procedures for the purification of mammalian viral reverse transcriptases. In the first procedure, a nucleic acid-free extract of Rauscher murine leukemia virus was applied to a column of phosphocellulose and the reverse transcriptase was eluted by a low concentration (50 microM) of polyguanylic acid. Polyadenylic acid and polyuridylic acid could not replace polyguanylic acid for the elution. In the second procedure, a polyuridylic acid-Sepharose column was substituted for phosphocellulose, and the elution was again achieved by polyguanylic acid. In the third affinity procedure, the reverse transcriptase in a nucleic acid-free viral extract was incubated in the cold with 50 microM polyguanylic acid and the complex was adsorbed onto a DEAE-cellulose column. After washing to remove uncomplexed and weakly complexed proteins, the reverse transcriptase was eluted in a concentrated form at 0.3 M NaCl with a recovery of greater than 70%. by polyacrylamide gel analysis in the presence of sodium dodecyl sulfate, the enzyme appeared to be nearly pure.

Chromatography, Affinity↗

PubMed1992

Fidelity of HIV-1 reverse transcriptase copying RNA in vitro.

The genomic hypervariation of human immunodeficiency virus 1 (HIV-1) could result from misincorporations by the viral reverse transcriptase. We developed an assay for reverse transcriptase fidelity during RNA-dependent as well as DNA-dependent DNA polymerization in vitro. A lacZ alpha RNA fragment transcribed by T3 RNA polymerase was used to mimic first-strand reverse transcription. The corresponding DNA template was used to examine errors by reverse transcriptase during second-strand DNA synthesis. With both templates, the mutations introduced by reverse transcriptase were identified by their mutant phenotypes in an M13 lacZ alpha-complementation assay. We found that the reverse transcriptase from human immunodeficiency virus 1 (HIV-1 RT) was less accurate than the reverse transcriptase from Moloney murine leukemia virus (MLV RT) or the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) on either RNA or DNA templates. The frequency of misincorporation by HIV-1 RT was 1 in 6900 nucleotides polymerized on the RNA template and 1 in 5900 on the DNA template. The error rates of MLV RT and Pol I on the RNA template were less than 1 in 28,000 and 37,000, respectively. The most frequent mutations produced by HIV-1 RT copying the RNA template were C----T transitions and G----T transversions resulting from misincorporation of dAMP.

Animals↗

PubMed2003

Hologram quantitative structure-activity relationships investigations of non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as TIBO, HEPT and dipyridodiazepinone are effective against HIV-1 RT. These NNRTIs are chemically and structurally diverse, but they all bind to a common allosteric site of HIV-1 RT. These inhibitors exhibit high potency, low cytotoxicity and produce few side effects. However, the emergency of drug-resistance viral strain has limited the therapeutic efficiency of the NNRTIs. Several different QSAR studies were reported to identify important structural features responsible for the inhibitory activity of these NNRTIs. In this study, hologram quantitative structure-activity relationships (HQSAR) was applied to three different data sets, 70 TIBO, 101 HEPT and 125 dipyridodiazepinone derivatives. Starting geometries of compounds were taken from available X-ray crystallographic data. Modification and full geometry optimization of all derivatives were performed, based on quantum chemical calculations at the HF/3-21G level of theory. All derived HQSAR models produce satisfying predictive ability and yield r(2)(cv) values ranging from 0.62-0.84. Moreover, it was also found that the quality of models enhances as the size of fragments increases. The obtained HQSAR results indicate the similarity of the interactions of these three different NNRTIs with the inhibition pocket of the enzyme. Comparisons of different QSAR methods on these NNRTIs data sets were also considered and it could be shown that HQSAR results yield superior predictive models than other 2D-QSAR approaches. In particular, the predictive ability of the models derived from dipyridodiazepinone analogues was significantly improved and apparently revealed differentiating structural requirements between WT and Y181C HIV RT inhibition. Additionally, the quality of QSAR models constructed by CoMFA and HQSAR methods are comparable and the interpretations of the models reinforce each other. It suggests an advantage of HQSAR as a useful tool in designing new potent inhibitors with enhanced HIV-1 RT inhibition activity, especially against mutant enzyme.

Anti-HIV Agents↗

PubMedDate not supplied

[Reverse transcriptase of the human immunodeficiency virus: cloning, expression in Escherichia coli, purification of the enzyme, and production of monoclonal antibodies].

To express HIV-1 reverse transcriptase in E. coli a number of genetic constructions containing reverse transcriptase and virus protease nucleotide sequences was obtained. The products of expression were characterized; monoclonal antibodies to reverse transcriptase were produced. The purification of reverse transcriptase was carried out. The substantial proteolysis of reverse transcriptase during purification was shown. The purified preparation is predominantly, an active protein with Mr 57 kDa. Some properties of this protein differed from the reverse transcriptase isolated from HIV.

Amino Acid Sequence↗

PubMed1997

De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence.

The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.

Base Composition↗

PubMed1996

Interaction of human immunodeficiency virus type 1 reverse transcriptase with primer tRNALys3 and affinity modification of the enzyme by tRNALys3 derivatives.

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV-1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non-complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70-80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity. In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo-first-order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Kd values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.

Animals↗