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PubMed1991

Equine infectious anemia virus and human immunodeficiency virus DNA synthesis in vitro: characterization of the endogenous reverse transcriptase reaction.

The endogenous reverse transcriptase reaction of equine infectious anemia virus (EIAV) has been studied, and conditions allowing synthesis of full-length minus-strand DNA have been determined. In contrast to results reported for other retroviruses, synthesis of EIAV full-length minus-strand DNA was not impaired by high concentrations of Nonidet P-40, a nonionic detergent used to make the virion envelope permeable. All components of the reaction were titrated for maximum synthesis of complete minus strands, and a time course under the standardized conditions was determined. Minor subgenomic bands were observed in some cases, and both the size and proportion varied with reaction conditions. Conditions established for full-length EIAV DNA synthesis also allowed full-genome-length human immunodeficiency virus type 1 DNA synthesis. The human immunodeficiency virus type 1 DNA product contained a greater proportion of reverse transcripts that were shorter than the complete virus genome. Also in contrast to EIAV, the endogenous synthesis of high-molecular-weight human immunodeficiency virus type 1 DNA was drastically reduced at Nonidet P-40 concentrations above 0.02%. These results indicated that a detergent-stable core is not a property shared by all lentiviruses. The EIAV virion synthetic machinery is unusually stable and provides a convenient system for further in vitro study of reverse transcription.

Acetates↗
PubMed1984

Affinity of reverse transcriptase for some polynucleotide inhibitors.

The relative affinity of avian myeloblastosis virus reverse transcriptase for (U)n and a series of (U)n analogs has been measured directly in solution under conditions previously used to demonstrate the inhibitory properties of these polynucleotides. The affinities were measured by electron spin resonance through a quantitative competition approach where the concentration of each polynucleotide required to compete with the macromolecular spin probe (ls4U,U)n for reverse transcriptase was observed. Using this approach the following relative affinities were determined: K(dUfl)n = 1.6K(dUz)n = 13K(dT)n = 20K(U)320-640 = 57K(dU)n = 167K(U)80 greater than 167K(Um)n These results show that the affinity of (U)n for reverse transcriptase is affected by modifying the (U)n matrix and by the molecular weight of (U)n. In addition, the effect of some factors such as Mg+2 and salt on the polynucleotide affinity for the enzyme was measured. The results show that the same binding, i.e., the fraction of saturation F as a function of the nanomoles of reverse transcriptase added, was observed in the presence or absence of Mg+2, whereas increasing the KCl concentration from 0.04 to 0.5M completely dissociates the polynucleotide X enzyme complex.

Avian Myeloblastosis Virus↗
PubMed1992

Pyridoxal-5'-phosphate inhibits the polymerase activity of a recombinant RNAase H-deficient mutant of HIV-1 reverse transcriptase.

We have investigated the ability of pyridoxal-5'-phosphate to inhibit a recombinant deletion mutant of human immunodeficiency virus type 1(HIV-1) reverse transcriptase (RT) which is missing the last 23 amino acids of the C-terminus. This mutant reverse transcriptase is characterized by normal polymerase activity as compared with full-length enzyme; however, it has no RNase H activity. Inhibition studies with pyridoxal-5'-phosphate showed several differences as compared with inhibition of full-length enzyme: (1) Inhibition of mutant reverse transcriptase was independent of divalent cation, (2) Either substrate alone could protect mutant reverse transcriptase from inactivation by pyridoxal-5'-phosphate, and (3) stoichiometry of pyridoxal-5'-phosphate binding to mutant reverse transcriptase was 2 mol/mol under the same conditions in which 1 mol/mol bound to full-length enzyme. Furthermore, in the presence of either substrate alone, the stoichiometry of pyridoxal-5'-phosphate binding to the mutant was reduced to 1 mol/mol. These results indicate that the second binding site for pyridoxal-5'-phosphate seen in the mutant reverse transcriptase is at or near the primer-template binding site of the enzyme. They also suggest that the RNase H domain of HIV RT plays a functional role in substrate binding at the polymerase domain.

DNA-Directed DNA Polymerase↗
PubMed1981

Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase.

A rapid assay for retroviral reverse transcriptase activity released into the culture medium by infected cells was developed. With the assay, 4,000 clonally infected cell lines could be tested in a few hours. We have adapted the assay for use as a screen for the detection of spontaneous viral mutants. Mutants of Moloney murine leukemia virus have been isolated which (i) produce a thermolabile reverse transcriptase, (ii) are temperature sensitive for release of enzyme activity, or (iii) can only productively infect cells already producing gag-related polypeptides. The assay has also been useful for the isolation of nonproducer cells infected with various replication-defective transforming viruses.

Culture Media↗
PubMed1990

Ribavirin is an inhibitor of human immunodeficiency virus reverse transcriptase.

Ribavirin inhibits the human immunodeficiency virus reverse transcriptase in an in vitro reaction. Ribavirin-5'-diphosphate was close to 40% more inhibitory than ribavirin-5'-triphosphate. Unphosphorylated ribavirin had a reduced, but detectable, effect as an inhibitor, compared with the phosphorylated forms. The compounds seem to have a direct effect on the viral polymerase, and no chain termination was observed in the presence of ribavirin-5'-triphosphate. Combination of any of the ribavirin derivatives tested with 3'-azido-3'-deoxythymidine (zidovudine)-5'-triphosphate resulted in an increase of its anti-human immunodeficiency virus reverse transcriptase activity in the in vitro assay.

Avian Myeloblastosis Virus↗
PubMed1992

Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein. The preparation of 23 murine monoclonal antibodies (mAb) against HIV-1 reverse transcriptase and their different effects on the enzyme are described. The interaction of purified mAbs with HIV-1 RT was demonstrated by enzyme-linked immunosorbent assay (ELISA), Western blots, and high performance liquid chromatography size exclusion chromatography. One of the antibodies also recognized recombinant HIV-2 RT. Antibody binding epitopes on HIV-1 RT were analyzed by immunoblotting using cyanogen bromide fragmented RT, C-terminally truncated mutants, and a peptide ELISA employing 15-mer synthetic overlapping peptides spanning nearly the complete polypeptide chain. The epitopes were mapped within three domains corresponding to amino acids 200-230, 300-428, and 528-560. Two mAbs show neutralizing properties on enzymatic functions of RT. One affects the polymerase activity and to a certain degree the RNase H activity of the enzyme, whereas the other inhibits the latter activity exclusively. mAb 28, which blocks the polymerase activity, interferes with the nucleotide binding region of RT, as shown by fluorescence spectroscopy using a labeled template/primer complex. By investigating the antibody effects on dimer formation of the heterodimeric enzyme, three domains corresponding to amino acids 230-300, 350-428, and residues around amino acid 540 involved in protein-protein interactions were localized.

Antibodies, Monoclonal↗
PubMed1990

Simian immunodeficiency virus reverse transcriptase. Purification and partial characterization.

Native reverse transcriptase from simian immunodeficiency virus was purified from virus with good recovery to near homogeneity. The optimum reaction conditions of the enzyme were determined with respect to divalent cations, pH and ionic strength. The enzyme was shown to possess both RNA-dependent and DNA-dependent DNA synthesis activity. In addition, we could demonstrate an associated RNase H activity. Employing novel assay conditions, activated DNA as a heteropolymeric substrate was used more efficiently than the homopolymeric substrate poly(rA).oligo(dT) which in turn was used twofold more effectively as the template primer than poly(dC).oligo(dG). Other homopolymeric substrates, including poly(rC).oligo(dG), were also tested but were found to be poorly used by the reverse transcriptase. The Miachaelis-Menten constants were determined for each of the four nucleotides needed to elongate a natural template primer. Simultaneously, using dideoxyadenosine triphosphate as nucleotide analogue, we could show that this compound acts as a competitive inhibitor with respect to dATP, whereas it acts as a non-competitive inhibitor with respect to the other nucleotides. Gel electrophoretic analysis showed the enzyme to consist of two polypeptides with apparent molecular masses of 64 and 48 kDa. Using activity gel electrophoresis, we were able to demonstrate that both subunits exhibit DNA synthesis activity.

Animals↗
PubMed1984

Purification of a specific inhibitor of reverse transcriptase from human placenta.

Human placental extracts contain a factor which specifically and reversibly inhibits the reverse transcriptase of mammalian retroviruses. This placental inhibitor has been partially purified and characterized. It elutes at 0.1-0.2 M phosphate on hydroxyapatite chromatography and can be further purified by phosphocellulose chromatography where it elutes at 0.4 M KCl. By these purification procedures, specific activities of 40-70,000 units of inhibitor per mg of protein were obtained. The size of the inhibitor is about 60-65,000 daltons as estimated by velocity sedimentation. The inhibitor purified by these techniques selectively inhibits the activity of purified reverse transcriptase from Rauscher murine leukemia virus and baboon endogenous virus. It is substantially less active against the reverse transcriptase of avian myeloblastosis virus. The specificity of this inhibitor for mammalian enzymes and particularly for the human placental reverse transcriptase suggests that it plays a role in the regulation of DNA synthesis in human placental development.

Animals↗
PubMed1977

Reverse transcriptase of foamy virus. Purification of the enzymes and immunological identification.

Reverse transcriptase from foamy virus, strain H4188 was estimated and purified. The enzyme has the following characteristics: 1. The reaction utilized preferentially oligo (dT) poly (rA) as a primer-template; however, the synthetic primer-template oligo (dT) poly (dA) could also be used to some extent. 2. The reaction utilized oligo (dG) poly (rC) as a primer-template with very low efficiency. 3. The crude virus preparation had a detectable endogenous reaction using the four deoxyribonucleotides for DNA polymerization. 4. The cation requirement for the enzyme reaction was much more biased for Mn++ than for Mg++ ions. 5. The molecular weight of the partially-purified enzyme was estimated to be about 80,000. Aggregates of 240,000 daltons were also seen. The activity of this enzyme was not inhibited by antisera against the reverse transcriptases of various type C RNA viruses, namely, feline endogenous leukemia virus, RD 114, Woolly simian sarcoma virus (SSV-1) and avian myeloblastosis virus (AMV). Antiserum against Rauscher leukemia virus (RLV) enzyme was marginally active against foamy virus enzyme, perhaps indicating a slight cross-reaction. The biochemical characteristics of foamy virus reverse transcriptase seemed to be very close to those of the type C RNA viruses, but the immunological reaction proved that the foamy virus reverse transcriptase was distinct from the others.

Epitopes↗
PubMedDate not supplied

Immunohistochemical evaluation of reverse transcriptase in breast carcinoma with polyclonal antibodies raised in rabbit.

In this study, the presence of reverse transcriptase in breast tumours was examined with immunoperoxidase staining using antibodies raised in rabbit against reverse transcriptase of Moloney murine leukemia virus and against reverse transcriptase of avian myeloblastosis virus. The specificity of such antibodies was investigated with ELISA and Western blotting techniques. Five cases of infiltrating ductal carcinomas were found positive with the immune serum anti-reverse transcriptase of Moloney murine leukemia virus on 28 studied infiltrating ductal carcinomas, 2 infiltrating lobular carcinomas and 2 fibroadenomas.

Adenofibroma↗
PubMed1990

Processing of the primer for plus strand DNA synthesis by human immunodeficiency virus 1 reverse transcriptase.

We have analyzed the processing of the RNA primer for (+) strand DNA synthesis by reverse transcriptase of the human immunodeficiency virus 1. To test for specific RNA cleavage and primer usage, we constructed a 99-base pair RNA-DNA hybrid containing the viral polypurine tract and flanking viral sequences. Although the RNase H activity of reverse transcriptase cleaves the RNA strand into multiple fragments, only two primers are extended in the presence of nucleoside triphosphates. The major RNA primer includes the entire polypurine tract except for the last adenosine and has the sequence 5'-UUUUAAAAGAAAAGGGGGG-3'. The minor primer has the same 3' end but is two nucleotides shorter. In a subsequent processing step reverse transcriptase releases the primer intact via a cleavage at the RNA-DNA junction. RNA cleavage, primer extension, and primer removal can take place in a single reaction. However, specificity does not require coupling of the three steps and is preserved in the individual reactions. The polypurine primer is generated and removed after its elongation in the absence of DNA synthesis. Furthermore, the polypurine primer is selected among the several RNA fragments available and extended by reverse transcriptase as well as by p51, a short form of reverse transcriptase lacking RNase H activity.

Base Sequence↗
PubMed1979

Characterization of the reverse transcriptase of a type C RNA virus produced by a human lymphoma cell line.

The reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) of the type C RNA virus produced by the human lymphoma cell line SU-DHL-1 was purified by ion-exchange chromatography of SU-DHL-1 culture fluids and repetitive affinity chromatography on poly(rC).agarose, as were the polymerases of several other type C viruses. The DHL-1 enzyme used template-primers at levels expected of a viral reverse transcriptase, and sodium dodecyl sulfate gel electrophoretic analysis of radioiodinated DHL-1 enzyme revealed a peak at a position corresponding to those of several other type C viral reverse transcriptases (namely, at 72,000-78,000 daltons). The purified enzyme was partially neutralized by antibodies specific for the reverse transcriptase of simian sarcoma virus. Two-dimensional analysis on thin-layer cellulose plates of tryptic hydrolysates of the radioiodinated enzymes of several viruses revealed that six peptides are common to the polymerases of simian sarcoma virus, gibbon ape leukemia virus, baboon endogenous virus, and the DHL-1 virus, and that two to four peptides are unique to each of these enzymes. The DHL-1 viral reverse transcriptase appears to be most closely related structurally to the enzymes of simian sarcoma virus, gibbon ape leukemia virus, and baboon endogenous virus. However, the DHL-1 viral enzyme differed from any one or combination of the other subhuman primate viral enzymes by virtue of its unique peptides. The implications of these findings with respect to the probable origin of the DHL-1 virus are discussed.

Cell Line↗
PubMed1989

Differential inhibitory effects of various herb extracts on the activities of reverse transcriptase and various deoxyribonucleic acid (DNA) polymerases.

Forty preparations of the extracts from 28 kinds of Asian herbs were tested for ability to inhibit the activities of murine retroviral reverse transcriptase and human deoxyribonucleic acid (DNA) polymerases. Among the 40 extracts, 35 inhibited reverse transcriptase activity and 29 inhibited DNA polymerase alpha activity. The inhibitory potencies of these extracts were expressed as the 50% inhibition concentrations (IC50), at which the enzyme activities were inhibited by 50%. Very strong inhibitions were observed with the extracts from Millettia pachycarpa (Leguminosae) and Mallotus apelta (Euphorbiaceae) as shown by their low IC50 values for reverse transcriptase (0.4-0.5 micrograms/ml) and DNA polymerase alpha (0.9-1.4 micrograms/ml). Enzyme kinetic analysis revealed that the mode of inhibition of reverse transcriptase by these two extracts was competitive with respect to the template.primer [poly(rA).oligo(dT)] and noncompetitive with respect to deoxythymidine triphosphate (dTTP) substrate. Besides reverse transcriptase and DNA polymerase alpha, DNA polymerase I and ribonucleic acid (RNA) polymerase from E. coli were inhibited by these two extracts. These results indicate that the herb extracts contain as yet unidentified substance(s) which inhibit the activities of reverse transcriptase and cellular DNA polymerases.

Humans↗
PubMed1985

Ty element transposition: reverse transcriptase and virus-like particles.

We have found reverse transcriptase activity and virus-like particles only in yeast cells that contain a galactose-promoted Ty element induced on galactose. The cofractionation of reverse transcriptase, genomic-length Ty RNA, and a Ty-specified protein antigen in a particulate fraction and the ability of this complex to synthesize specifically a product that is homologous to the entire Ty suggest that reverse transcription of Ty RNA takes place in the particle. The absence of appreciable levels of reverse transcriptase and particles in uninduced cells despite the presence of at least 35 copies of chromosomal Ty elements suggest that some of these elements may be defective. The numerous virus-like particles visible in thin sections of Ty transposition-induced cells appear not to be infectious. These particles resemble the intracisternal A-type particles of the mouse and copia particles of Drosophila. The results support the idea that Ty elements and retroviruses share a common origin.

Antigens, Fungal↗
PubMed1991

[Polymerase and immunologic activity of reverse transcriptase of the HIV-1 virus, isolated from Escherichia coli].

The recombinant reverse transcriptase of HIV-1 virus has been isolated from Escherichia coli cells transformed by the plasmid pRT40 DNA. The 103 Kd protein produced by these cells is shown to be processed to proteins with lower molecular masses by the reverse transcriptases own protease activity as well as Escherichia coli proteases. The resulting 103-66 Kd proteins possess the polymerase activity while 51 Kd and smaller proteins are lacking the activity. The 66 and 51 Kd reverse transcriptase fragments demonstrate the positive immunological reaction with the human blood serum from the people possessing antibodies to HIV-1 virus. The recombinant reverse transcriptase of HIV-1 produced by Escherichia coli cells is shown to be useful in AIDS diagnosis in humans.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase.

Succinylfluorescein-labeled dideoxyTTP has been used as a substrate for reverse transcriptase from HIV-1. On addition to the 3'-end of a primer molecule, there is a reduction of fluorescence yield of a factor of ca. 4. Release of a fluorescent DNA/DNA primer/template duplex from its complex with reverse transcriptase results in a reduction of fluorescence by a further factor of 2. The fluorescent nucleotide is incorporated somewhat less efficiently than 3'-azidoTMP and TMP, which show similar incorporation kinetics. Fluorescent chain-terminated primers have been used to investigate the interaction of normal and chain-terminated primer/template complexes with reverse transcriptase. The dissociation constant of a 36/18-mer was 0.65 nM, whereas that of the same complex after the addition of the fluorescent chain-terminating nucleotide to the primer was 3 nM at 25 degrees C. The rate of dissociation of the latter complex from the enzyme was 0.04 s-1. This was decreased by a factor of ca. 10 at high concentrations (greater than 200 microM) of the nucleotide triphosphate complementary to the next position of the template. The results obtained suggest that potent inhibition of reverse transcriptase activity in in vitro assays results from formation of a slowly dissociating complex between the enzyme and chain-terminated primer/template complexes. However, arguments are presented that lead to the conclusion that this is not the mode of inhibition in cells invaded by HIV. At the prevailing relative concentrations in this situation, chain termination resulting in incomplete transcription is likely to be the major factor.

Binding, Competitive↗
PubMed1991

Avian myeloblastosis virus reverse transcriptase. Effect of glycerol on its hydrodynamic properties.

Although reverse transcriptase has been the subject of intensive investigation, minimal information is available regarding the physical properties of the enzyme. The basic hydrodynamic properties of avian myeloblastosis virus reverse transcriptase in solution were measured by both sedimentation velocity and equilibrium measurements in two buffer systems. In a 0.3 M potassium phosphate buffer system, pH 7.8, the enzyme sedimented as a homogenous particle with a sedimentation coefficient of (7.1 +/- 0.3) S with a weight-average molecular weight, Mw, of (1.52 +/- 0.05) x 10(5). Since the enzyme consists of an alpha and beta subunit of equal molar ratio with Mw of 6.3 x 10(4) and 9.4 x 10(4), respectively, it was concluded that the enzyme exists as an alpha beta heterodimer in this buffer system. In a Tris buffer system, pH 7.9, containing 0.46 M NaCl and 4% glycerol, the native enzyme also sedimented as a homogeneous particle with an apparent sedimentation coefficient of (10.1 +/- 0.5) S, without considering the effect of glycerol on solvent-protein interaction. Based on the results of Gekko and Timasheff (Gekko, K., and Timasheff, S. N. (1981) Biochemistry 20, 4667-4676) and the polarity of the enzyme, it was estimated that there is significant solvent-protein interaction even at 4% glycerol leading to a value of -0.06 g/g in the preferential solvent interaction parameter. When the solvent effect was taken into consideration, the value for s020,w increased from 10.1 to 11.9 S, implying that the native enzyme dimerizes in the presence of 4% glycerol. The combined results of gel filtration and sedimentation velocity showed that the dimerization of the enzyme to form (alpha beta)2 is favored at 20 degrees C with the alpha beta form predominating at 4 degrees C. The secondary structure of the reverse transcriptase was measured by circular dichroism. Results showed that the enzyme consists of (16 +/- 2)% alpha-helix, (24 +/- 2)% beta-sheet, (24 +/- 2)% beta-turn, and (36 +/- 4)% undefined structures.

Avian Myeloblastosis Virus↗
PubMed1990

Differential inhibition of the activities of reverse transcriptase and various cellular DNA polymerases by a traditional Kampo drug, sho-saiko-to.

A traditional Kampo drug, Sho-saiko-to, composed of several herb extracts, differentially inhibited the activities of reverse transcriptase and human cellular DNA polymerase alpha and beta. Reverse transcriptases from murine leukemia virus and human immunodeficiency virus were inhibited by over 80% and 50%, respectively, in the presence of 100 micrograms/ml Sho-saiko-to, whereas DNA polymerase alpha was much less sensitive to inhibition by this drug than were the reverse transcriptases. DNA polymerase gamma was not inhibited by this drug at concentrations of up to 500 micrograms/ml. Only DNA polymerase beta was moderately inhibited by Sho-saiko-to. Thus, it has been shown that the inhibition by Sho-saiko-to is relatively specific for reverse transcriptase and that the drug contains as yet unidentified inhibitory substance(s) for reverse transcriptase.

Animals↗