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PubMed1996

Preoperative reverse transcriptase polymerase chain reaction for prostate specific antigen predicts treatment failure following radical prostatectomy.

PURPOSE: We previously demonstrated than an enhanced reverse transcriptase-polymerase chain reaction assay for prostate specific antigen (PSA) can predict final pathological stage in radical prostatectomy patients. The potential role of the assay in predicting serum PSA recurrence after radical prostatectomy was explored. MATERIALS AND METHODS: We evaluated 100 radical prostatectomy candidates by reverse transcriptase polymerase chain reaction preoperatively, and status was compared to serum PSA, Gleason score and final pathological results. Potential surgical failure was defined as tumor at the surgical margin or extending into the seminal vesicle. Patients were monitored postoperatively by serum PSA every 4 months. Kaplan-Meier analysis was used to evaluate the correlation between reverse transcriptase polymerase chain reaction and disease recurrence, defined as a PSA of 0.2 ng/ml. or greater. RESULTS: Enhanced reverse transcriptase polymerase chain reaction for PSA had a stronger correlation with potential surgical failure than preoperative serum PSA or Gleason score (relative risks 15.2, 5.9 and 3.2, respectively). The correlation between these modalities and PSA recurrence was evaluated during a mean followup of 13.6 months (range 5 to 26). Of 36 patients with positive reverse transcriptase polymerase chain reactions 9 had failure by PSA compared to 3 of 64 (4.7%) with negative polymerase chain reactions (p<0.0286). The relative risk for failure by reverse transcriptase polymerase chain reaction was 3.6. Gleason score and serum PSA had higher correlations with postoperative PSA elevations (relative risk 13.2 and 7.6, respectively). A Cox regression analysis model demonstrated that reverse transcriptase polymerase chain reaction for PSA can be used in conjunction with Gleason score and provides statistically significant risk information. CONCLUSIONS: Enhanced reverse transcriptase polymerase chain reaction for PSA is a statistically significant predictor of potential failure by pathological analysis and of disease recurrence by PSA. Longer followup data are required to define further the role of the assay in the management of patients with prostate cancer.

Aged↗
PubMed1974

Reverse transcriptase in normal rhesus monkey placenta.

Particles with the morphology of type C virus have been identified from primate placentas by electron microscopy. A reverse transcriptase (RNA-dependent DNA polymerase) was isolated and purified from microsomal pellets of two fresh placentas of rhesus monkeys in the early stages of gestation. This enzyme was biochemically similar yet immunologically distinct from the reverse transcriptases of known tumorigenic type C RNA viruses isolated from primates, but was immunologically related to a reverse transcriptase isolated from a type C virus obtained from normal baboon placenta. These particles may represent endogenous viruses and may function in the transfer of genetic information during embryogenesis.

Animals↗
PubMed1989

Characterization of a reverse transcriptase activity associated with retrovirus-like particles in a Drosophila cell line.

The properties of an endogenous RNA-dependent DNA polymerase (reverse transcriptase) associated with retrovirus-like particles (VLP) in a Drosophila melanogaster cell line were characterized. The enzyme requires a monovalent and a divalent cation, a sulfhydril-reducing agent and the four deoxynucleosides triphosphates for a maximal activity. The reaction was enhanced by a detergent and was sensitive to a DNA-A-free RNA-A indicating that the enzymatic activity was indeed associated with VLP which contain intrinsic RNA. The maximal incorporation of the labelled deoxynucleotide was observed at 25 degrees C in the presence of Mn2+. The enzyme responded well to exogenous template-primers in the similar way to that of retroviral reverse transcriptase and the use of several inhibitors confirms the presence of a real reverse transcriptase activity associated with virus-like particles in drosophila cells.

Animals↗
PubMed1995

Resistance to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives is generated by mutations at multiple sites in the HIV-1 reverse transcriptase.

Virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross-resistance evaluation indicated that the two drug-resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives. The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400-resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross-resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400. Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (> 20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds.

Antiviral Agents↗
PubMed1999

Reversion of a human immunodeficiency virus type 1 matrix mutation affecting Gag membrane binding, endogenous reverse transcriptase activity, and virus infectivity.

We previously characterized mutations in the human immunodeficiency virus type 1 matrix (MA) protein that displayed reduced infectivity in single-round assays, defects in the stable synthesis of viral DNA in infected cells, and impaired endogenous reverse transcriptase activity. The mutants, which contained substitutions in a highly conserved Leu at MA amino acid 20, also increased binding of Gag to membrane. To elucidate further the role of MA in the virus replication cycle, we have characterized a viral revertant of an amino acid 20 mutant (20LK). The revertant virus, which replicates with essentially wild-type kinetics in H9 cells, contains second-site compensatory changes at MA amino acids 73 (E-->K) and 82 (A-->T), while retaining the original 20LK mutation. Single-cycle infectivity assays, performed with luciferase-expressing viruses, show that the 20LK/73EK/82AT triple mutant displays markedly improved infectivity relative to the original 20LK mutant. The stable synthesis of viral DNA in infected cells is also significantly increased compared with that of 20LK DNA. Furthermore, activity of revertant virions in endogenous reverse transcriptase assays is restored to near-wild-type-levels. Interestingly, although 20LK/73EK/82AT reverses the defects in replication kinetics, postentry events, and endogenous reverse transcriptase activity induced by the 20LK mutation, the reversion does not affect the 20LK-imposed increase in Gag membrane binding. Mutants containing single and double amino acid substitutions were constructed, and their growth kinetics were examined. Only virus containing all three changes (20LK/73EK/82AT) grew with significantly accelerated kinetics; 73EK, 73EK/82AT, and 20LK/82AT mutants displayed pronounced defects in virus particle production. Viral core-like complexes were isolated by sucrose density gradient centrifugation of detergent-treated virions. Intriguingly, the protein composition of wild-type and mutant detergent-resistant complexes differed markedly. In wild-type and 20LK complexes, MA was removed following detergent solubilization of the viral membrane. In contrast, in revertant preparations, the majority of MA cosedimented with the detergent-resistant complex. These results suggest that the 20LK/73EK/82AT mutations induced a significant alteration in MA-MA or MA-core interactions.

Amino Acid Sequence↗
PubMed2003

HIV-1 reverse transcriptase and protease resistance mutations selected during 16-72 weeks of therapy in isolates from antiretroviral therapy-experienced patients receiving abacavir/efavirenz/amprenavir in the CNA2007 study.

OBJECTIVE: To determine HIV-1 reverse transcriptase (RT) and protease (PRO) mutations selected in isolates from antiretroviral therapy (ART)-experienced patients receiving an efavirenz/abacavir/amprenavir salvage regimen. METHODS: Open-label, single arm of abacavir, 300 mg twice daily, amprenavir, 1200 mg twice daily and efavirenz, 600 mg once daily, in ART-experienced patients of which 42% were non-nucleoside reverse transcriptase inhibitor-naive. The virology population examined consisted of all patients who took at least 16 weeks of study drugs (n=74). Plasma population sequencing was carried out at baseline and last time point at which patients were still taking the three study drugs + other ART. The median follow-up was 48 weeks (range week 16-72). RESULTS: Baseline (n=73) and on-therapy (n=49) genotypes were obtained. By 48 weeks, 51% of isolates had > or = 3 non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations. NNRTI mutations selected on therapy were K103N (51%), substitutions at position 190 (17/49, 35%): G to A (n=11) / S (n=4) / E (n=1) and T (n=1); L100I (37%) and V1081 (20%) mutations. P225H was not observed in this study. L100I and G190A/S/E/T mutations were rarely detected in the same viral population and baseline Y181C favoured the G190 mutations (OR=8.9, P<0.001), rather than the L100I. The NRTI mutations selected were in accordance with abacavir known resistance profile, no new TAMs were observed, new L74V or I mutations developed in 39 and 16% of isolates, respectively, however, new M184V mutations were only detected in isolates from two patients, one of whom had added lamivudine + didanosine. M184V was common at baseline (55%) and maintained in 22/27 (81%) isolates (five of these 22 added lamivudine or didanosine, or both). The PRO mutations selected were in accordance with the distinct resistance profile of amprenavir compared with other protease inhibitors. Mutations D30N, G48V, N88D/S, L90M and 154V were de-selected, and mutations I50V, I or V to 54M/L, I84V, M46I/L, L33F, I47V as well mutations at position 10 were observed in 20/49 (41%) isolates. CONCLUSION: Prior NNRTI and NRTI therapy influences the pathway of resistance to efavirenz. In this study, the prevalence of mutations selected by efavirenz were different from those described in less ART-experienced patients. Baseline Y181C was associated with the development of mutations at position 190, but not L100I or K103N. In this patient population, abacavir with efavirenz preferentially selected for L74V but not for thymidine analogue mutations. M184V was rarely selected and was maintained in only 77% of patients who did not add lamivudine or didanosine. Finally, amprenavir-specific mutations were selected in the background of other primary protease inhibitor mutations, confirming the distinct resistance profile of amprenavir.

Alkynes↗
PubMed1980

Purification of reverse transcriptase from avian retroviruses using affinity chromatography on heparin-sepharose.

Reverse transcriptase from Rous sarcoma virus and avian myeloblastosis virus was purified by a rapid two-step procedure using chromatography on phosphocellulose and heparin-Sepharose. The resulting enzyme was homogeneous, had a high specific activity and was free of contaminating nucleases. This procedure has been adapted to small-scale preparation of enzyme from mutant virus containing thermolabile reverse transcriptase, and is equally suitable for large-scale enzyme purification.

Avian Leukosis Virus↗
PubMed1992

The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis.

Hepatitis B viruses (hepadnaviruses) replicate their DNA genomes by reverse transcription of an RNA intermediate. Efforts to examine the biochemical mechanism for viral DNA synthesis have been hampered by the failure to solubilize the reverse transcriptase from virions and to express the polymerase in heterologous systems in an enzymatically active form. Here, we demonstrate that the polymerase of a hepadnavirus synthesized in an in vitro translation reaction exhibits reverse transcriptase activity. Furthermore, our results show that the polymerase acts as a primer for DNA synthesis and remains covalently linked to nascent DNA, a feature that is not known to exist in any other RNA-directed DNA polymerases. Priming of DNA synthesis requires viral RNA but occurs independently of other viral components. The ability to express the hepadnavirus reverse transcriptase in an enzymatically active form will allow detailed biochemical and functional analyses of this complex enzyme, and may facilitate the identification of inhibitors required for antiviral therapy.

Base Sequence↗
PubMed2003

Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection.

An accurate, sensitive and specific reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors zalcitabine, lamivudine, didanosine, stavudine, zidovudine, and abacavir with the non-nucleoside reverse transcriptase inhibitor nevirapine in human blood plasma is described. The new Polarity dC C(18) silica column used in this method provides better resolution and peak shape than all other columns tested. Also, four different ultraviolet wavelengths were used for accurate and specific quantitation of the analytes. The method was validated over the range of 10-10000 ng/ml for all analytes except zalcitabine (10-5000 ng/ml). This method is accurate (average accuracies of three different concentrations ranged from 97.2 to 105%), and precise (within- and between-day precision measures ranged from 0.5 to 5.1% and 0.5 to 5.6%, respectively), and is currently being used for determination of plasma drug concentrations in our laboratory.

Chromatography, High Pressure Liquid↗
PubMed2002

Synthesis of alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors with non-identical aromatic rings.

The existing methods for the synthesis of alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors proceed from symmetrical benzophenones and therefore result in products with identical aromatic rings. New methods have therefore been devised for the preparation of stereochemically defined ADAMs with non-identical aromatic rings. The new routes rely on palladium-catalyzed reactions, including Sonogashira, Suzuki, Stille, and hydroarylation methodology. Several of the new ADAMs inhibited the cytopathic effect of HIV-1 in cell culture and HIV-1 reverse transcriptase at submicromolar concentrations.

Biochemistry↗
PubMed1995

Reverse transcriptase reads through a 2'-5'linkage and a 2'-thiophosphate in a template.

Avian myeloblastosis virus and Maloney murine leukemia virus RNase H-reverse transcriptases pause when they encounter a 2'-5' linkage or a 2'-thiophosphate in their template RNAs, but eventually read through these backbone modifications. Both reverse transcriptases pause after the 2'-5' linkage but before the 2'-thiophosphate. These results suggest that in the absence of precise information concerning the behavior of a given reverse transcriptase with respect to a particular lesion or modification, caution should be exercised in the interpretation of primer extension data that is being used to determine the existence of, or map the position of, a crosslink, site of chemical modification or non-standard linkage in an RNA template.

Avian Myeloblastosis Virus↗
PubMed2006

Evidence that creation of invasion sites determines the rate of strand transfer mediated by HIV-1 reverse transcriptase.

Strand transfer during reverse transcription can produce genetic recombination in human immunodeficiency virus type 1 (HIV-1) when two genomic RNAs, that are not identical, are co-packaged in the virus. Strand transfer was measured in vitro, in reactions involving primer switching from a donor to acceptor RNA template. The transfer product appeared with much slower kinetics than full-length synthesis on the donor template. The goal of this study was to learn more about the transfer mechanism by defining the steps that limit its rate. We previously proposed transfer to include the steps of acceptor invasion, hybrid propagation, terminus transfer, and re-initiation of synthesis on the acceptor template. Unexpectedly, with our templates increasing acceptor concentration increased the transfer efficiency but had no effect on the rate of transfer. Templates with a short region of homology limiting hybrid propagation exhibited a slow accumulation of transfer products, suggesting that for tested long homology templates hybrid propagation was not rate limiting. Substituting a DNA acceptor and adding Klenow polymerase accelerated re-initiation and extension exclusively on the DNA acceptor. This lead to a small rate increase due to faster extension on the acceptor, suggesting re-initiation of synthesis on the tested RNA acceptors was not rate limiting. A substrate was designed in which the 5' end of the primer was single stranded, and complimentary to the acceptor, i.e. having a pre-made invasion site. With this substrate, increasing concentrations of acceptor increased the rate of transfer. Together these data suggest that RNase H cleavage, and dissociation of RNA fragments creating an invasion site was rate limiting on most tested templates. When an accessible invasion site was present, acceptor interaction at that site influence the rate.

DNA Primers↗
PubMed1993

Potent and highly selective human immunodeficiency virus type 1 (HIV-1) inhibition by a series of alpha-anilinophenylacetamide derivatives targeted at HIV-1 reverse transcriptase.

In vitro evaluation of a large chemical library of pharmacologically acceptable prototype compounds in a high-capacity, cellular-based screening system has led to the discovery of another family of human immunodeficiency virus type 1 (HIV-1) inhibitors. Through optimization of a lead compound, several alpha-anilinophenylacetamide (alpha-APA) derivatives have been identified that inhibit the replication of several HIV-1 strains (IIIB/LAI, RF, NDK, MN, HE) in a variety of host cell types at concentrations that are 10,000- to 100,000-fold lower than their cytotoxic concentrations. The IC50 of the alpha-APA derivative R 89439 for HIV-1 cytopathicity in MT-4 cells was 13 nM. The median 90% inhibitory concentration (IC90) in a variety of host cells was 50-100 nM. Although these alpha-APA derivatives are active against a tetrahydroimidazo [4,5,1-jk][1,4]benzodiazepin-2(1H)-thione-(TIBO)-resistant HIV-1 strain, they do not inhibit replication of HIV-2 (strains ROD and EHO) or simian immunodeficiency virus (strains Mac251, mndGB1, and agm3). An HIV-1 strain containing the Tyr181-->Cys mutation in the reverse transcriptase region displayed reduced sensitivity. alpha-APA derivative R 89439 inhibited virion and recombinant reverse transcriptase of HIV-1 but did not inhibit that of HIV-2. Reverse transcriptase inhibition depended upon the template/primer used. The relatively uncomplicated synthesis of R 89439, its potent anti-HIV-1 activity, and its favorable pharmacokinetic profile make R 89439 a good candidate for clinical studies.

Acetamides↗
PubMed2001

Synthesis, biological activity, pharmacokinetic properties and molecular modelling studies of novel 1H,3H-oxazolo[3,4-a]benzimidazoles: non-nucleoside HIV-1 reverse transcriptase inhibitors.

New 1H,3H-oxazolo[3,4-albenzimidazoles (OBZs) were synthesized as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) to extend the structure-activity relationships observed for an early series of related 1H,3H-thiazolo[3,4-a]benzimidazole derivatives (TBZs). The new compounds showed inhibitory activity against the replication of various HIV-1 strains, including NNRTI-resistant strains. Testing of a representative OBZ derivative in an HPLC assay on biological fluids, indicated that the sulphur substitution appreciably improved the metabolic stability of the TBZ compound. In addition, molecular modelling studies demonstrated that OBZs, TBZs and other NNRTIs have similar structural properties, that is a butterfly-like conformation, which is a key structural requirement for reverse transcriptase inhibition.

Animals↗
PubMed1994

Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase.

The accepted model of retroviral reverse transcription includes a circular DNA intermediate which requires strand displacement synthesis for linearization and creation of an integration-competent, long terminal repeat-flanked DNA product. We have used an in vitro model of this last step of reverse transcription to examine the role of the viral enzyme, reverse transcriptase (RT), in displacement synthesis. We show that Moloney murine leukemia virus RT possesses an activity which allows for displacement synthesis through a minimum of 1,334 bp of duplex DNA--an extent much greater than that required during in vivo reverse transcription and over 25-fold greater than has been previously demonstrated for a viral RT. RT does not function as a helicase in the classical sense but appears to closely couple duplex DNA melting with synthesis-driven translocation of the enzyme. In the absence of synthesis, the unwound region created by a primer-positioned RT appears to be no greater than 2 bp and does not advance along the template. Additionally, RT does not utilize ATP or any deoxynucleoside triphosphate not directly encoded by the template strand to catalyze processive duplex unwinding at a nick; nor does binding of the enzyme unwind duplex DNA in the absence of a 3' terminus. The approximate maximum chain elongation rate during strand displacement synthesis by Moloney murine leukemia virus RT falls between 0.73 and 1.5 nucleotides per s at 37 degrees C. The RNase H activity of RT does not appear to play a role in displacement synthesis; however, a 181-amino-acid C-terminal truncation of RT displays a dramatically reduced ability to catalyze synthesis through duplex DNA.

Base Sequence↗
PubMed1993

Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem.

During processive DNA synthesis in vitro, the human immunoefficiency virus, type 1 (HIV-1) reverse transcriptase encounters template nucleotide positions at which continued synthesis is difficult. At these positions, the enzyme has a relatively high probability of dissociating from the template, and product molecules of corresponding length accumulate as the incubation proceeds. These positions, which are known as termination sites, could be associated with template secondary structures in some cases, but many termination sites appear to be template sequence-related rather than secondary structure-related. Mechanisms producing these blocks in processive DNA synthesis are not well understood. In this study, to examine further the effects of template sequence on termination, we engineered selected single-base changes in the M13mp2 template, and we found that such changes can influence termination. Several general trends emerged from the study. First, strong termination sites rarely correspond to dATP as the "incoming" substrate opposite template T. Second, the sequence of the template-primer stem is more important for termination than the sequence of the single-stranded template ahead of the primer. Thus, we note the phenomenon of action at a distance: changing sequence at one nucleotide position in the template-primer stem alters termination at other positions, a few nucleotides distant at the primer 3' end. A and C as template bases in the template-primer stem have opposite effects. A is the strongest terminator residue, and C is the weakest terminator residue, followed by G. Since termination sites are produced by reverse transcriptase dissociation from the template-primer, the results suggest that the HIV-1 reverse transcriptase has properties reminiscent of a sequence-specific double-stranded DNA-binding protein in that its binding mechanism can distinguish both base residues and positions in the double-stranded DNA template-primer stem.

Base Sequence↗
PubMed1998

Expression of mouse telomerase reverse transcriptase during development, differentiation and proliferation.

We have identified the mouse telomerase reverse transcriptase component (mTERT) and demonstrate both substantial sequence homology to the human ortholog (hTERT), and the presence of reverse transcriptase and telomerase specific motifs. Furthermore, we show functional interchangeability with hTERT in in vitro telomerase reconstitution experiments, as mTERT produces strong telomerase activity in combination with the human telomerase RNA component hTR. The mouse TERT is widely expressed at low levels in adult tissues, with greatest abundance during embryogenesis and in adult thymus and intestine. The mTERT component mRNA levels were regulated during both differentiation and proliferation, while mTR levels remained constant throughout both processes. Comparison of mTERT and mTR levels to telomerase activity indicates that mTERT expression is more tightly linked to the regulation of telomerase activity during these processes than is mTR. In contrast to the situation in human cell cultures, mTERT transcript levels are present at readily detectable levels in primary cultured cells and are not upregulated following crisis. The widespread expression of mTERT in primary cells and mouse tissues could explain the increased frequency of spontaneous immortalization of mouse cells in culture and tumorigenesis in vivo.

3T3 Cells↗
PubMed1988

3'-end labeling of DNA fragments by AMV-reverse transcriptase.

The efficiency on 3'-end labeling of DNA fragments was compared between Klenow fragment of DNA polymerase I from E. coli and AMV-reverse transcriptase. In the case of Klenow fragment, the exonuclease activity of this enzyme rapidly removed the labeled dNMP incorporated in 3'-end of DNA fragment by the polymerase activity of it. AMV-reverse transcriptase caused no decrease of the labeled dNMP incorporated by the polymerase activity because it has no exonuclease activity. Therefore, AMV-reverse transcriptase is easier to use than Klenow fragment for labeling the 3'-end of DNA fragment.

Avian Myeloblastosis Virus↗