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PubMed1990

Site-directed mutagenesis of Moloney murine leukemia virus reverse transcriptase. Demonstration of lysine 103 in the nucleotide binding site.

Lys103 and Lys421 of Moloney murine leukemia virus reverse transcriptase have been implicated in the dNTP binding function as judged by their reactivity to a substrate binding site-directed reagent, pyridoxal 5'-phosphate (Basu, A., Nanduri, V. B., Gerard, G. F., and Modak, M. J. (1988) J. Biol. Chem. 263, 1648-1653). To assess the true catalytic importance of the individual lysine residues in Moloney murine leukemia virus reverse transcriptase, we mutated Lys103 and Lys421 to leucine and alanine, respectively. Analysis of the mutant enzymes revealed that mutation at the 103 position had a drastic effect on the DNA polymerase activity whereas the 421 mutation had no effect. Both mutants exhibited normal RNase H activity as well as the ability to bind to RNA or DNA templates as judged by UV-mediated cross-linking of the enzyme to the template primers. The enzyme with mutation at codon 421 (Lys----Ala) exhibited properties that were indistinguishable from the wild type with respect to its mode of catalysis, i.e. preference of template primer and divalent metal ion, RNA- or DNA-dependent DNA polymerase activity, RNase H activity, and the processive mode of DNA synthesis. These observations suggest that only Lys103 and not Lys421 is the catalytically important residue that is involved in the binding of substrate dNTP in Moloney murine leukemia virus reverse transcriptase.

Base Sequence↗

PubMed2003

Functional reverse transcriptase encoded by the human LINE-1 from baculovirus-infected insect cells.

The human LINE-1 ORF2, which encodes reverse transcriptase, was inserted into a baculovirus shuttle vector and expressed in Sf 21 cells. An immunoreactive polypeptide (149kDa) synthesized by infected cells had reverse transcriptase activity. A procedure for purification of functional ORF2 protein from insect cells was developed. The enzyme was purified with good recovery to near homogeneity and retained stable DNA polymerase activity. The optimum reaction conditions of the enzyme were determined with respect to salts, pH, and temperature. Substrate specificities and divalent cation requirements were investigated. The recombinant enzyme had a 3-fold preference for Mg2+ over Mn2+ for reverse transcriptase activity on poly(rA).oligo(dT)(12). As for DNA synthesis, the recombinant ORF2 protein was found to possess both RNA-dependent and DNA-dependent DNA polymerase activities.

Animals↗

PubMed1997

Molecular staging of prostate cancer. III. Effects of cystoscopy and needle biopsy on the enhanced reverse transcriptase polymerase chain reaction assay.

PURPOSE: We examined the effects of prostatic manipulations, including flexible cystoscopy and transrectal needle biopsy, on the enhanced reverse transcriptase polymerase chain reaction assay in 57 men. MATERIALS AND METHODS: The reverse transcriptase polymerase chain reaction assay was performed on 25 patients with clinically localized stages T1 to T2cN0M0 prostate cancer before and 30 minutes after cystoscopy. In addition, blood specimens from 32 patients with elevated serum prostate specific antigen and/or abnormal digital rectal examinations were tested immediately before and at 30 minutes after transrectal ultrasound guided prostate needle biopsy. RESULTS: We detected no difference between polymerase chain reaction results obtained immediately before and 30 minutes after cystoscopy in 25 men. Transrectal needle biopsy had no effect on the polymerase chain reaction results in 30 of 32 men. However, 2 men had positive reactions on post-biopsy specimens only. Pathological results of the biopsy revealed benign prostatic hyperplasia and prostatic intraepithelial neoplasia, respectively, in these 2 men. CONCLUSIONS: We conclude that cystoscopy has no clinically significant effect on the reverse transcriptase polymerase chain reaction assay. However, prostatic needle biopsy may cause a positive polymerase chain reaction for PSA in the immediate post-biopsy period.

Biopsy, Needle↗

PubMed1992

HIV inhibitors targeted at the reverse transcriptase.

HIV inhibitors targeted at the virus-associated reverse transcriptase (RT) can be divided into two groups, depending on whether they are targeted at the substrate or nonsubstrate binding site. To the first group belong the 2',3'-dideoxynucleosides (i.e., DDC, DDI), 3'-azido-2',3'-dideoxynucleosides (i.e., AZT), 3'-fluoro-2',3'-dideoxynucleosides (i.e., FLT), 2',3'-didehydro-2',3'-dideoxynucleosides (i.e., D4C, D4T) and carbocyclic derivatives thereof (i.e., carbovir), 2'-fluoro-ara-2',3'-dideoxynucleosides, 1,3-dioxolane derivatives (i.e., 2',3'-dideoxyl-3'-thiacytidine), oxetanocin analogues and carbocyclic derivatives thereof (i.e., cyclobut-G) and the 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives. These compounds need to be phosphorylated intracellularly to their triphosphate forms before they act as competitive inhibitors or alternate substrates (chain terminators) of HIV RT. The second group includes the tetrahydro-imidazo[4,5,l-jk][1,4]-benzodiazepin-2(1H)one (TIBO), 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT), dipyrido[3,2-b:2',3'-e]-[1,4]diazepin-6-one (nevirapine) and pyridin-2(1H)one derivatives, which interact as such, noncompetitively, with a specific allosteric binding site of HIV-1 RT. Compounds belonging to the two different groups may give rise to synergism which combined, and, likewise, viral resistance to the compounds may arise through different mutations, depending on the nature of the compounds and the group to which they belong.

Adenine↗

PubMed1996

Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase.

OBJECTIVE: To measure the extent of HIV resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) within the context of monotherapy and to assess the presence of the M184V substitution in the case of 3TC-resistant viruses. Whether the success of 3TC in clinical trials could be due, in part, to an increase in the fidelity of HIV reverse transcriptase conferred by the M184V substitution was also considered. METHODS: Two separate monotherapy studies were evaluated, one involving adults with CD4 counts > or = 300 x 10(6)/l, and the second involving children, some of whom had received antiretroviral treatment previously, while others were drug naive. Peripheral blood and plasma samples were collected regularly, and HIV isolation and determinations of drug median inhibitory concentration values were performed using umbilical cord mononuclear cells as targets. Amplification of the 184 mutation was performed by the polymerase chain reaction, using specific primer pairs. Fidelity determinations using purified, recombinant HIV reverse transcriptase derived from either wild-type virus or viruses that contained the 184V substitution were performed. RESULTS: Phenotypic resistance was detected in almost all subjects at times ranging from 8-20 weeks after initiation of therapy. The 184V substitution was usually detected prior to the occurrence of phenotypic resistance to 3TC. Fidelity determinations revealed that the 184V substitution conferred an approximately 5- to 10-fold increase in HIV reverse transcriptase fidelity. In addition, titres of patient sera tested for their ability to neutralize autologous sequential viral isolates were stabilized in patients receiving 3TC therapy as opposed to other drugs. CONCLUSIONS: Resistance to 3TC developed in virtually all subjects treated with this drug, and was associated with the appearance of an M184V mutation in HIV reverse transcriptase. The clinical benefit of 3TC therapy may be attributable in part to selection of viruses that are less able to replicate and mutate than the wild types.

Adult↗

PubMed1984

Interactions between avian myeloblastosis reverse transcriptase and tRNATrp. Mapping of complexed tRNA with chemicals and nucleases.

The interactions between beef tRNATrp with avian myeloblastosis reverse transcriptase have been studied by statistical chemical modifications of phosphate (ethylnitrosourea) and cytidine (dimethyl sulfate) residues, as well as by digestion of complexed tRNA by Cobra venom nuclease and Neurospora crassa endonuclease. Results with nucleases and chemicals show that reverse transcriptase interacts preferentially with the D arm, the anticodon stem and the T psi stem. All these regions are located in the outside of the L-shaped structure of tRNA. This domain of interaction is different to that reported previously in the complex of beef tRNA with the cognate aminoacyl-tRNA synthetase (M. Garret et al.; Eur. J. Biochem. In press). Avian reverse transcriptase destabilizes the region of tRNA where most of the tertiary interactions maintaining the structure of tRNA are located.

Alkylation↗

PubMed2004

Resistance to dual nucleoside reverse-transcriptase inhibitors in children infected with HIV clade A/E.

The prevalence of nucleoside reverse-transcriptase inhibitor (NRTI) mutations was determined among 95 human immunodeficiency virus-infected Thai children who were treated with dual nucleoside reverse-transcriptase inhibitors. Almost all children had resistance to at least 1 NRTI, and approximately half of the children had resistance to multiple NRTIs. Cross-resistance to stavudine and azidothymidine was universal.

Child↗

PubMed1998

Telomerase reverse transcriptase genes identified in Tetrahymena thermophila and Oxytricha trifallax.

Telomerase reverse transcriptase (TERT) has been identified as the catalytic subunit of the chromosome end-replicating enzyme in Euplotes, yeasts, and mammals. However, it was not reported among the protein components of purified Tetrahymena telomerase, the first telomerase identified and the most thoroughly studied. It therefore seemed possible that Tetrahymena used an alternative telomerase that lacked a TERT protein. We now report the cloning and sequencing of a Tetrahymena thermophila gene whose encoded protein has the properties expected for a TERT, including large size (133 kDa), basicity (calculated pI = 10.0), and reverse transcriptase sequence motifs with telomerase-specific features. The expression of mRNA from the Tetrahymena TERT gene increases dramatically at 2-5 h after conjugation, preceding de novo addition of telomeres to macronuclear DNA molecules. We also report the cloning and sequencing of the ortholog from Oxytricha trifallax. The Oxytricha macronuclear TERT gene has no introns, whereas that of Tetrahymena has 18 introns. Sequence comparisons reveal a new amino acid sequence motif (CP), conserved among the ciliated protozoan TERTs, and allow refinement of previously identified motifs. A phylogenetic tree of the known TERTs follows the phylogeny of the organisms in which they are found, consistent with an ancient origin rather than recent transposition. The conservation of TERTs among eukaryotes supports the model that telomerase has a conserved core (TERT plus the RNA subunit), with other subunits of the holoenzyme being more variable among species.

Amino Acid Sequence↗

PubMed1987

Antiretroviral activity in a marine red alga: reverse transcriptase inhibition by an aqueous extract of Schizymenia pacifica.

An aqueous extract from the marine red alga, Schizymenia pacifica has been tested in a cell free system for its effect on reverse transcriptase from avian retrovirus (avian myeloblastosis virus), and mammalian retrovirus (Rauscher murine leukemia virus). The extract inhibited reverse transcriptase from both these retroviruses but showed almost no effect, if any, on the activity of cellular DNA polymerase alpha and RNA polymerase II in vitro. Consequently it is unlikely to have an adverse effect on the growth of cultured cell. The inhibitory activity of the extract was stable over a relatively wide pH range (pH 1-11) and was not lost after pronase digestion. Inhibitory activity of the extract was lost after boiling at 100 degrees C in 0.67 N HCl, and after treatment with 100 mM NaIO4. The active principle in the extract has an apparent molecular weight in excess of 100,000 daltons. This new reverse transcriptase inhibitor is probably a polysaccharide.

Antiviral Agents↗

PubMed1978

Reverse transcriptase activity in extracts of human prostatic tissues.

The extracts of human prostatic tissue specimens contain oncornavirus-like reverse transcriptase activity. This activity was isolated by banding the tissue extract in an equilibrium sucrose density gradient followed by phosphocellulose chromatography of the lysate of the material banding at a density of 1.14--1.20 g/cc. It was characterised by its utilisation of poly (Cm) as a template and its inhibition by selective inhibitors of viral reverse transcriptase. Human prostatic tissues of three histo-pathologic types--normal, hyperplastic and adenocarcinoma--were examined. One out of four normal, five out of six hyperplastic, and two out of two adenocarcinoma specimens displayed virus-like reverse transcriptase activity.

Adenocarcinoma↗

PubMed1991

The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase.

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.

Amino Acid Sequence↗

PubMed1991

Reverse transcriptase from human immunodeficiency virus: a single template-primer binding site serves two physically separable catalytic functions.

The binding of substrates to recombinant reverse transcriptase from human immunodeficiency virus (HIV) and the natural enzyme from avian myeloblastosis virus (AMV) has been examined by analyzing both the ribonuclease H and the RNA-dependent DNA polymerase activities. With 3'-end-labeled globin mRNA hybridized to (dT)15 as the substrate in the ribonuclease H reaction, the enzymes partially deadenylated the mRNA in a distributive manner. Under these conditions, there was a rapid initial burst followed by a prolonged, but much slower, steady-state rate. The biphasic reaction made possible determinations of kinetic constants as follows: values for Km, KD, and kcat were, respectively, 27 nM, 11 nM, and 5 x 10(-3) s-1 for the HIV enzyme and 30 nM, 9 nM, and 5 x 10(-3) s-1, respectively, for the avian enzyme. These constants were used to derive other parameters: The rate of association of the template-primer with reverse transcriptase was approximately 2 x 10(5) M-1 s-1, and the rate of dissociation was approximately 2 x 10(-3) s-1, regardless of the source of the enzyme. The rate of release of the product was essentially equivalent to the value of kcat indicated above for each of the enzymes. The polymerase reaction was evaluated under processive conditions of synthesis; values of Km and kcat of approximately 6 nM and approximately 2.5 s-1, respectively, for the human enzyme, and approximately 10 nM and approximately 2 s-1, respectively, for the avian enzyme were observed. The interaction of substrates with HIV reverse transcriptase was characterized further with the aid of ribonucleoside-vanadyl complexes. These complexes inhibited the polymerase and ribonuclease H activities of the enzyme competitively with respect to globin mRNA.(dT)15. Values of Ki ranging from 1 to 3 mM were obtained. With respect to deoxyribonucleoside triphosphate substrates in the polymerase reaction, mixed inhibition was observed. Deoxyribonucleoside triphosphates had no effect on kinetic parameters governing the ribonuclease H activity of the HIV enzyme but apparently facilitated the formation of active enzyme. These data fit a model in which one template-primer binding site serves both the polymerase and the ribonuclease H catalytic sites.

Avian Myeloblastosis Virus↗

PubMed2003

Human telomerase reverse transcriptase motifs required for elongation of a telomeric substrate.

The reverse transcriptase telomerase copies an internal RNA template to synthesize telomeric simple-sequence repeats. In the cellular context, telomerase must elongate its few intended substrates (authentic chromosome ends) without spurious activity on other potential substrates (chromosome ends created by damage, repair, or recombination). Many mechanisms have been proposed to account for the biological substrate specificity of telomerase, with most models focusing on protein-protein interactions between telomerase and telomeric chromatin. Telomerase activity assays testing the elongation of model oligonucleotide substrates have revealed that in addition to hybridization with the RNA template, optimal DNA substrates also engage telomerase protein-based interaction sites. The physiological significance of these non-template interaction sites has not been established. We used in vivo reconstitution to assemble telomerase enzymes with variant telomerase reverse transcriptase proteins. Several telomerase enzyme variants retained a wild-type level of catalytic function in vitro when assayed using an artificial sequence substrate but exhibited reduced activity on a more physiological telomeric-sequence substrate. Telomerases that demonstrated this defect in telomeric substrate usage in vitro also failed to support telomere length maintenance in vivo. Our findings suggest that non-template interactions of the telomerase ribonucleoprotein with telomeric DNA play a critical role in supporting telomerase function on its appropriate cellular substrates.

Amino Acid Motifs↗

PubMed1999

Nitric oxide inhibits the HIV-1 reverse transcriptase activity.

Nitric oxide (NO) is a polypotent regulatory molecule involved in a variety of activities, such as the modulation of the catalytic activity of cysteine-containing enzymes. The present study reports the modulation of the HIV-1 reverse transcriptase activity by NO, released by the NO-donors 3, 3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18), (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), 3-morpholinosydnonimine (SIN-1), 4-(phenylsulfonyl)-3-((2-(dimethylamino) ethyl)thio)furoxan oxalate (SNO-102), and sodium nitroprusside (SNP). NO inhibits dose-dependently the HIV-1 reverse transcriptase activity, likely due to oxidation of Cys residue(s). Present results, representing a new insight into the modulation mechanism of the HIV-1 reverse transcriptase activity, may be relevant to develop new strategies for inhibition of HIV-1 replication.

Catalysis↗

PubMed2001

Factors affecting the dimerization of the p66 form of HIV-1 reverse transcriptase.

The association and dissociation of the homodimeric p66/p66 form of HIV-1 reverse transcriptase were investigated. The effects on the dimerization process of different salt concentrations, pH and the presence of a template/primer and nucleotide substrates were monitored by measuring polymerase activity and analytical size-exclusion HPLC. At submicromolar concentrations of enzyme and physiological salt concentrations, most of the enzyme exists in the inactive monomeric form. Increasing NaCl concentration from 0.05 to 1 M decreased the equilibrium dissociation constant from 2.0 to 0.34 microM. Analysis of the kinetics of the dimerization process indicated it followed a two-step mechanism, with rapid initial association of the two subunits to form an inactive homodimer followed by a slow isomerization step rendering the active enzyme form. The presence of poly(rA)/dT(20) decreased the equilibrium dissociation constant of the homodimer about 30-fold, while the addition of 5 microM dTTP had no effect. The kinetics of the process showed that the template/primer favored dimerization by binding to the inactive homodimer and promoting its isomerization to the active form. These results were confirmed by analyzing the reverse reaction, i.e. the dissociation of the enzyme, by dilution in a low-ionic-strength buffer. The results suggest that binding of immature HIV-1 reverse transcriptase to its natural template/primer may be relevant in both the dimerization process and the selection of its natural primer.

Base Sequence↗

PubMed1999

The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity.

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.

Amino Acid Sequence↗

PubMed1979

Suramin: a potent inhibitor of the reverse transcriptase of RNA tumor viruses.

Suramin--a well-known antitrypanosomal agent--was found to exert a strong inhibitory effect on the RNA-directed DNA polymerase (reverse transcriptase) activity of several oncornaviruses such as Moloney murine leukemia virus, murine Rauscher leukemia viruses, Moloney murine sarcoma virus and avian myeloblastosis virus. Inhibition of enzyme activity was obtained with both endogenous viral RNA and (A)n . oligo(dT) as the template-primer. Suramin effected a 50% inhibition of the reverse transcriptase activity of oncornaviruses at a concentration range of 0.1--1 microgram/ml. In this aspect it compared favorably to ethidium bromide, another trypanocide drug which is considered as one of the most powerful inhibitors of oncornaviral DNA polymerases. The inhibition of reverse transcriptase activity by suramin was competitive with the template-primer, (A)n . oligo(dT), suggesting that the drug may interact with the template-primer binding site of the enzyme.

Avian Myeloblastosis Virus↗

PubMed1998

Quantification of thyroid hormone receptor isoforms, 9-cis retinoic acid receptor gamma, and nuclear receptor co-repressor by reverse-transcriptase PCR in maturing and adult skeletal muscles of rat.

Quantitative competitive reverse-transcriptase polymerase chain reaction (qcRT-PCR) was established for determining absolute molecule numbers of the thyroid hormone receptor (T3R) isoforms T3Ralpha1, T3Ralpha2, T3Rbeta1, and the 9-cis retinoic acid receptor gamma (RXRgamma) in developing and adult fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles of rat. Expression levels of the nuclear receptor co-repressor (NCoR) were measured in the same muscles because responses to thyroid hormones during muscle maturation might not only depend on the expression levels of the various receptors but might also be modulated by changes in the expression of NCoR. The qcRT-PCR method was based on the addition of known amounts of homologous competitor RNAs to the reverse transcriptase (RT) reaction. We show that all nuclear receptors under study were expressed in fast and slow muscles. Transcript numbers of T3Rbeta1, which was the most abundant isoform, were higher in SOL than in EDL during all developmental stages. The mRNAs for T3Ralpha1, T3Ralpha2, RXRgamma and the NCoR displayed molecule numbers in similar ranges, but were differentially expressed. T3Ralpha1 mRNA increased in SOL during postnatal development, while T3Ralpha2 mRNA initially decreased, then increased to adult levels. Conversely, pronounced decreases were observed for T3Ralpha1 (10-fold) and T3Ralpha2 (28-fold) mRNAs in the EDL muscle during postnatal maturation. RXRgamma mRNA was 10-fold downregulated during EDL maturation, but unaltered in maturing SOL. NCoR transcript number displayed only minor changes in both muscles.

Aging↗