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PubMed1998

Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity.

Disseminated neoplasia (DN), a proliferative cell disorder of the circulatory system of bivalves, was first reported in oysters in 1969. Since that time, the disease has been determined to be transmissible through water-borne exposure, but the etiological agent has not been unequivocally identified. In order to determine if a viral agent, possibly a retrovirus, could be the causative agent of DN, transmission experiments were performed, using both a cell-free filtrate and a sucrose gradient-purified preparation of a cell-free filtrate of DN positive materials. Additionally, a PCR-enhanced reverse transcriptase assay was used to determine if reverse transcriptase was present in tissues or hemolymph from DN positive soft shell clams Mya arenaria. DN was transmitted to healthy clams by injection with whole DN cells, but not with cell-free filtrates prepared from either tissues from DN positive clams, or DN cells. The cell-free preparations from DN-positive tissues and hemolymph having high levels of DN cells in circulation exhibited positive reactions in the PCR-enhanced reverse transcriptase assay. Cell-free preparations of of hemolymph from clams having low levels of DN (<0.1% of cells abnormal), hemocytes from normal soft shell clams, and normal soft shell clam tissues did not produce a positive reaction in the PCR enhanced reverse transcriptase assay.

Animals↗
PubMed1988

The accuracy of reverse transcriptase from HIV-1.

A study was conducted to determine the fidelity of DNA synthesis catalyzed in vitro by the reverse transcriptase from a human immunodeficiency virus type 1 (HIV-1). Like other retroviral reverse transcriptases, the HIV-1 enzyme does not correct errors by exonucleolytic proofreading. Measurements with M13mp2-based fidelity assays indicated that the HIV-1 enzyme, isolated either from virus particles or from Escherichia coli cells infected with a plasmid expressing the cloned gene, was exceptionally inaccurate, having an average error rate per detectable nucleotide incorporated of 1/1700. It was, in fact, the least accurate reverse transcriptase described to date, one-tenth as accurate as the polymerases isolated from avian myeloblastosis or murine leukemia viruses, which have average error rates of approximately 1/17,000 and approximately 1/30,000, respectively. DNA sequence analyses of mutations generated by HIV-1 polymerase showed that base substitution, addition, and deletion errors were all produced. Certain template positions were mutational hotspots where the error rate could be as high as 1 per 70 polymerized nucleotides. The data are consistent with the notion that the exceptional diversity of the HIV-1 genome results from error-prone reverse transcription.

Avian Myeloblastosis Virus↗
PubMed1988

Enzyme activity gel analysis of human immunodeficiency virus reverse transcriptase.

An activity gel analysis was performed in order to examine the catalytically active component of human immunodeficiency virus (HIV) reverse transcriptase in purified enzyme preparations and HIV-infected cell extracts. Immunoaffinity purified HIV reverse transcriptase contains two proteins with molecular weights 66,000 and 51,000 in approximately equal proportions. After denaturing polyacrylamide gel electrophoresis and removal of sodium dodecyl sulfate, the p66 component of reverse transcriptase was sufficient for both DNA- and RNA-directed DNA synthesis. No DNA synthetic activity of p51 was observed. Recovery of p66 catalytic activity was approximately 10% that of DNA polymerase beta, and the density of the autoradiographic band corresponding to p66 was linear with enzyme concentration. No additional HIV-specific DNA polymerases besides p66 were observed in HIV-infected H9 cell extracts.

DNA Polymerase I↗
PubMed1980

Biochemical and immunological properties of the DNA polymerase and RNAase H activities of purified feline leukemia virus reverse transcriptase.

Feline leukemia virus DNA polymerase was purified by ion-exchange and nucleic acid affinity chromatographies. The enzyme consists of a single polypeptide chain of mol. wt. approx. 72,000 as determined by both glycerol density gradient centrifugation and SDS-polyacrylamide gel electrophoresis. The preferred divalent cation for DNA synthesis is Mn2+ on a variety of template-primers, and its optimum concentration appears to be significantly lower than reported results of other mammalian type-C viral enzymes. The purified enzyme also contained RNAase H activity. Both DNA polymerase and RNAase H activities appear to reside on the same molecule as demonstrated by the copurification of both activities through various purification steps. The divalent cation requirement for maximum activity of RNAase H is also similar to that of the DNA polymerase. RNAase H without detectable polymerase activity was generated by a limited chymotrypsin digestion of the purified reverse transcriptase. This RNAase H activity was inhibited equally effectively as RNAase H in the intact reverse transcriptase by antisera prepared against reverse transcriptase of feline leukemia virus. These results indicate that the RNAase H catalytic activity of reverse transcriptase is distinct from the polymerase portion of the molecule. Since the RNAase H activity appears to be more stable, the measurement of RNAase H activity with a proper antibody might be useful for assaying tumor cells for the presence of the viral enzyme.

Animals↗
PubMed1994

Selection of high-affinity RNA ligands to reverse transcriptase: inhibition of cDNA synthesis and RNase H activity.

Specific, high-affinity RNA ligands to avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases were isolated from a combinatorial RNA library using the SELEX (systematic evolution of ligands by exponential enrichment) procedure. The selected RNA ligands bound their respective reverse transcriptases with approximately nanomolar dissociation constants. The ligands did not exhibit primary sequence conservation from selections against different target enzymes. Moreover, the selected ligands competed with the binding of template/primer complex and inhibited both the RNA-dependent DNA polymerase and the RNase H activities of the cognate reverse transcriptase. SELEX can yield both high-affinity and high-specificity oligonucleotide antagonists against specific members of a protein family.

Avian Myeloblastosis Virus↗
PubMedDate not supplied

[Substrate properties of dinucleoside 5',5"-oligophosphates in the reactions catalyzed by HIV reverse transcriptase, E. coli DNA polymerase I, and E. coli RNA polymerase].

Dinucleoside 5',5"-oligophosphates dNp4dN', dTp3rA, dTp4rA, and dTp5rA were synthesized and evaluated as substrates for HIV reverse transcriptase, the Klenow fragment of E. coli DNA polymerase I, and E. coli RNA polymerase. A model of the HIV reverse transcriptase interaction with the dinucleoside oligophosphates was proposed and experimentally confirmed. The kinetic constants of some of the synthesized compounds for the HIV reverse transcriptase were determined.

DNA Polymerase I↗
PubMed1999

Conformational study of the HIV-1 reverse transcriptase inhibitor 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT).

The conformations of the HIV-1 reverse transcriptase inhibitor 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) are calculated by semiempirical and mainly by ab initio methods in order to estimate the potential for the rotation around the carbon sulfur single bond. The results are compared to X-ray structures of HEPT associated to the HIV-1 reverse transcriptase. The NMR spectra of the compound are calculated to obtain some information about its structure in solution. The structure of HEPT in the complex is analysed to study the intermolecular interactions between the inhibitor and the surrounding protein, which determine the geometry of the inhibition complex.

Anti-HIV Agents↗
PubMed2005

Simultaneous determination of the HIV nucleoside analogue reverse transcriptase inhibitors lamivudine, didanosine, stavudine, zidovudine and abacavir in human plasma by reversed phase high performance liquid chromatography.

A reversed phase high performance liquid chromatography method was developed for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine, didanosine, stavudine, zidovudine and abacavir in plasma. The method involved solid-phase extraction with Oasis MAX cartridges from plasma, followed by high performance liquid chromatography with a SymmetryShield RP 18 column and ultraviolet detection set at a wavelength of 260 nm. The assay was validated over the concentration range of 0.015-5 mg/l for all five NRTIs. The average accuracies for the assay were 92-102%, inter- and intra-day coefficients of variation (CV) were <2.5% and extraction recoveries were higher than 97%. This method proved to be simple, accurate and precise, and is currently in use in our laboratory for the quantitative analysis of NRTIs in plasma.

Anti-HIV Agents↗
PubMed2003

Solution structure of the RNase H domain of the HIV-1 reverse transcriptase in the presence of magnesium.

This paper presents the first solution structure of the RNase H domain of HIV-1 reverse transcriptase (RT) determined by NMR methods. The solution conditions in this study were at physiological pH in the presence of Mg(2+). An investigation of the dependence of the (1)H-(15)N HSQC spectrum of the RNase H domain on [Mg(2+)] indicates that Mg(2+) produces significant, global effects on the amide chemical shifts, implying that divalent metal ion binding is important for stabilizing the structure of the isolated domain in solution. Analysis of amide shift data as a function of MgCl(2) concentration using either a single- or two-site binding model indicated that the latter provided a significantly improved fit, with the K(D) for site A = 2.7-3.2 mM and K(D) for site B approximately 35 mM, calculated on the assumption that site A is already occupied. Resonances of the [U-(13)C,(15)N]RNase H domain, measured at pH 6.8, in 80 mM MgCl(2), were assigned and NOESY data collected in order to determine the structure. Assignment of the NOESY spectra using the ARIA program resulted in a high-resolution structure for residues 6-114 which was similar to the crystal structure of the isolated domain,. The data were insufficient to define a compact structure for the C-terminal residues after 114. Residues I134-L138 located at the C-terminus are highly disordered and give rise to relatively sharp and intense amide resonances, while the amide resonances for the segment from E124 to A132 appear to be largely absent and are presumably subject to significant exchange broadening between different conformational states. Comparisons with crystal structure data for the full reverse transcriptase molecule indicate that the corresponding region is absent in nearly all of the crystal structures determined for the P2(1)2(1)2(1) space group, while these residues adopt an alpha-helix in structures determined for other symmetry groups. This structural heterogeneity indicates that significant conformational variability exists for this segment of the full reverse transcriptase enzyme as well, and the structure of the C-terminal peptide can be selected or deselected, depending on crystallization conditions. This analysis, along with the structural characterization contained herein, challenges the previous paradigm that the dynamic behavior of the isolated RNase H domain differs substantially from the behavior in the intact enzyme. The poor Mg(2+) binding and conformational flexibility of residues located near the active site indicate that substrate binding is a precondition for metal ion binding and for selecting the active site conformation of the RNase H domain.

Binding Sites↗
PubMed2000

Telomerase activity reconstituted in vitro with purified human telomerase reverse transcriptase and human telomerase RNA component.

Telomerase is a specialized reverse transcriptase that catalyzes elongation of the telomeric tandem repeat, TTAGGG, by addition of this sequence to the ends of existing telomeres. Human telomerase reverse transcriptase (hTERT) has been identified as a catalytic enzyme involved in telomere elongation that requires telomerase RNA, human telomerase RNA component (hTR), as an RNA template. We established a new method to express and purify soluble insect-expressed recombinant hTERT. The partially purified FLAG-hTERT retained the catalytic activity of telomerase in a complementation assay in vitro to exhibit telomerase activity in telomerase-negative TIG3 cell extract and in a reconstitution assay with FLAG-hTERT and purified hTR in vitro. FLAG-hTERT (D712A) with a mutation in the VDV motif exhibited no telomerase activity, confirming the authentic catalytic activity of FLAG-hTERT. The reconstituted complex of FLAG-hTERT and hTR in vitro was detected by electrophoretic mobility shift assay, and its activity was stimulated by more than 30-fold by TIG3 cell extract. This suggested that some cellular component(s) in the extract facilitated the reconstituted telomerase activity in vitro. Geldanamycin had no effect on the reconstituted activity but partially reduced the stimulated activity of the reconstituted telomerase by the TIG3 cell extract, suggesting that Hsp90 may contribute to the stimulatory effect of the cellular components.

Benzoquinones↗
PubMed1989

Inhibition of Moloney murine leukemia virus reverse transcriptase by alpha-anomeric oligonucleotides.

After parallel hybridization to complementary template RNA, alpha-anomeric oligonucleotides are not primers for Moloney murine leukemia virus reverse transcriptase. As can be expected they are competitors of classical primer oligonucleotides (beta-anomeric). They therefore inhibit the RNA dependent DNA polymerase activity of Moloney murine leukemia virus reverse transcriptase with either homopolymeric or heteropolymeric substrates. Non complementary alpha-oligonucleotides display no inhibitory activity. alpha-Oligonucleotides are therefore potential candidates for inhibition of retroviral reverse transcriptases by interference with the primer binding sites.

Base Sequence↗
PubMed1999

Docking experiments in the flexible non-nucleoside inhibitor binding pocket of HIV-1 reverse transcriptase.

Docking experiments were undertaken using a number of published crystal structures of HIV-1 reverse transcriptase complexes with various non-nucleoside inhibitors. The docking method was validated by successfully docking each ligand, in the conformation found in the crystal structure of the complex with the enzyme, back into its binding pocket in the right orientation and position. Each ligand was then subjected to conformational searching and a database of unique low-energy conformations of all ligands established. Docking this database into each of the reverse transcriptase binding pockets showed that all inhibitors could be fitted into each different pocket, without alteration of the pocket geometry. This contradicts findings from earlier docking investigations and implies that the conformation of the binding pocket in each different complex is conserved sufficiently to allow particular uniform ligand binding modes. The inhibitor conformations selected by this docking process are mostly the same as the one the ligand adopts in its own pocket and the selected conformations and orientations exhibit an impressive degree of similarity in the arrangement of their steric and electronic features. A correlation has also been observed between inhibitor flexibility and tightness of fit into the pockets with the more flexible inhibitors achieving a tighter fit and thus fewer favourable orientations upon docking.

Binding Sites↗
PubMed2000

Multiple sites in HIV-1 reverse transcriptase associated with virological response to combination therapy.

OBJECTIVE: To determine whether analysis of sequence variation in reverse transcriptase at baseline can explain differences in response to combination antiretroviral therapy. METHODS: Amino acid sequences of reverse transcriptase obtained from baseline isolates from 55 patients included in a trial of zidovudine and didanosine versus zidovudine/didanosine/nevirapine (ACTG241) were analysed. Simple and multiple linear regression were used to determine the relationship between numbers and identity of mutations at baseline and virological response after 8 and 48 weeks. RESULTS: Numbers of baseline zidovudine resistance mutations were predictive of short-term response (week 8). Amino acid identity at position 215 explained > 20% of the variation in response at week 8, but less at week 48. Multiple regression identified the combinations: 215 + 44 and 41 + 202, each of which explained about 30% of the variation in week 8 response. A model incorporating amino acids 214 + 215 + 60 + 202 + baseline viral load explained > 40% of the variation in response at week 48. Unexpectedly, the mutant combination 601 + 215Y/F responded threefold better than 60V + 215Y/F over 48 weeks. CONCLUSIONS: Use of clinical data to analyse virological response to combination therapy has revealed effects of baseline amino acid mutations at sites not previously identified as being important in antiretroviral resistance. Predictors of long-term responses were different from those involved in the short term and may require more complex analysis.

Acquired Immunodeficiency Syndrome↗
PubMed1995

The intracellular phosphorylation of (-)-2'-deoxy-3'-thiacytidine (3TC) and the incorporation of 3TC 5'-monophosphate into DNA by HIV-1 reverse transcriptase and human DNA polymerase gamma.

(-)-2'-deoxy-3'-thiacytidine (3TC) has been shown to be a potent, selective inhibitor of HIV replication in vitro, which requires phosphorylation to its 5'-triphosphate for antiviral activity. The intracellular concentration of 3TC 5'-triphosphate in phytohaemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBL) shows a linear dependence on the extracellular concentration of 3TC up to an extracellular 3TC concentration of 10 microM. At this extracellular concentration of 3TC, the resulting intracellular concentration of 3TC 5'-triphosphate is 5 microM. This value is similar to the inhibition constant (Ki) values for the competitive inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and human DNA polymerases (10-16 microM) by 3TC 5'-triphosphate. Since the concentration of 3TC producing 90% inhibition (IC90) of HIV replication in PBLs has been reported to be 76 nM, the antiviral activity of 3TC requires intracellular concentrations of 3TC 5'-triphosphate, which would result in very little inhibition of reverse transcriptase if its sole mode of action was competitive inhibition. This apparent discrepency may be explained by the ability of 3TC 5'-triphosphate to act as a substrate for reverse transcriptase. Primer extension assays have shown that 3TC 5'-triphosphate is a substrate for HIV-1 reverse transcriptase and DNA polymerase gamma, resulting in the incorporation of 3TC 5'-monophosphate into DNA. In the case of DNA polymerase gamma, the product of this reaction (i.e. double-stranded DNA with 3TC 5'-monophosphate incorporated at the 3'-terminus of the primer strand) is also a substrate for the 3'-5' exonuclease activity of this enzyme. This may explain the low levels of mitochondrial toxicity observed with 3TC.

Base Sequence↗
PubMed1998

Actinomycin D inhibition of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase and nucleocapsid protein.

Actinomycin D was found to be a potent inhibitor of HIV-1 reverse transcriptase catalyzed DNA strand transfer reactions. Using an oligonucleotide model system, actinomycin D inhibition of DNA strand transfer was examined to elucidate the mechanism of inhibition and further define the mechanism of DNA strand transfer. Our results show that actinomycin D inhibits HIV-1 reverse transcriptase catalyzed DNA strand transfer without inhibiting RNA-dependent or DNA-dependent DNA polymerase activity. Actinomycin D was found to strongly inhibit annealing of a primary DNA product to the DNA acceptor template, preventing the formation of a key reaction intermediate. The HIV-1 nucleocapsid protein has been shown to participate in catalytic events during reverse transcription including DNA strand transfer. Recombinant nucleocapsid protein was used in conjunction with actinomycin D in this model system to investigate how NC may participate in the mechanism of inhibition by actinomycin D and in DNA strand transfer. The inclusion of nucleocapsid protein was found to partially relieve both DNA annealing and strand transfer inhibition caused by actinomycin D. This study suggests a potential new mechanism for inhibiting retroviral replication by preventing the formation of replication intermediates.

Catalysis↗
PubMed2002

Functional analysis of the C-terminal extension of telomerase reverse transcriptase. A putative "thumb" domain.

Telomerase is an RNA-protein complex responsible for the extension of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to reverse transcriptases (RTs) encoded by retroviruses and retroelements. The mechanisms of telomerase may therefore be similar to those of the conventional reverse transcriptases. In this report, we explore potential similarity between these two classes of proteins in a region with no evident sequence similarity. Previous analysis has implicated a C-terminal domain of retroviral RTs (known as the "thumb" domain) in template-primer binding and in processivity control. The equivalent region of TERTs, although similar to one another, does not exhibit significant sequence homology to retroviral RTs. However, we found that removal of this region of yeast TERT similarly resulted in a decrease in the stability of telomerase-DNA complex and in the processivity of telomerase-mediated nucleotide addition. Moreover, the C-terminal domain of TERT exhibits a nucleic acid binding activity when recombinantly expressed and purified. Finally, amino acid substitutions of conserved residues in this region of TERT were found to impair telomerase activity and processivity. We suggest that mechanistic similarity between telomerase and retroviral RTs may extend beyond the regions with apparent sequence similarity.

Animals↗
PubMed1988

Duck hepatitis B virus: DNA polymerase and reverse transcriptase activities of replicative complexes isolated from liver and their inhibition in vitro.

The duck hepatitis B virus (DHBV)-associated activities of reverse transcriptase and DNA polymerase and their inhibition in vitro were studied. Replicative complexes (RCs) were isolated from DHBV-infected liver by gel chromatography followed by sucrose gradient centrifugation. The RCs were detected by dot blot hybridization, using radiolabeled cloned DHBV DNA as a probe, and by the incorporation of 32P-TTP in the presence of dATP, dCTP, dGTP, and Mg2+ (endogenous DNA polymerase activity). The endogenous DNA polymerase activity associated with RCs was further studied using exogenous templates: reverse transcriptase and DNA polymerase activities were demonstrated using as substrates 32P-TTP and poly(rA) p(dT)12 or poly(dA) p(dT)12-18, respectively. Both activities were biochemically characterized. Their inhibition by various antiviral agents was studied in vitro: actinomycin D, ara-ATP, aphidicolin, suramin, chloroquin, and phosphonoformate. Among these, suramin, chloroquin, phosphonoformate, and ara-ATP were shown to be potent inhibitors of viral reverse transcriptase and DNA polymerase. Studies are now in progress to establish their antiviral activity in vivo.

Animals↗
PubMed2001

Two independent regions of human telomerase reverse transcriptase are important for its oligomerization and telomerase activity.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, contains motifs conserved among reverse transcriptases. Several nucleic acid-dependent polymerases that share a "fingers, palm, and thumb substructure" were shown to oligomerize. Here we demonstrate that hTERT also has this ability using partially purified recombinant hTERTs and mammalian cells co-expressing differently tagged hTERTs. Human template RNA (hTR), by contrast, has no effect on the structural oligomerization of hTERTs. Therefore, hTERT has an intrinsic ability of oligomerization in the absence of hTR. We identified two separate regions as essential for the oligomerization. The regions, amino acids 301-538 (amino-terminal region) and amino acids 914-928 (carboxyl-terminal region), are outside the fingers and palm substructure covering motif T to D and interact with each other in vivo. A substituted mutant of hTERT, hTERT-D712A-V713I, which was reported as a dominant negative form of hTERT, bound to the wild-type hTERT and inhibited its telomerase activity transiently expressed in telomerase-negative finite normal human fibroblast. The truncated forms of hTERT containing the binding region to the wild-type hTERT partially inhibited the telomerase activity, probably by preventing the wild-type hTERT from forming an oligomer. Taken together, the oligomerization of hTERT is an important step for telomerase activity.

Amino Acid Motifs↗