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PubMed2001

Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in myxoid liposarcomas using LightCycler real-time quantitative reverse transcriptase-polymerase chain reaction.

We describe a convenient, nonradioactive reverse transcription--polymerase chain reaktion (RT-PCR) method for the rapid and accurate quantitative detection of the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) mRNA. The LightCycler TeloTAGGG hTERT Quantification Kit (Roche Molecular Biochemicals) was designed to be used for the highly sensitive and quantitative detection of hTERT mRNA relative to the house-keeping gene porphobilinogen deaminase (PBGD). As a tumor progression model, we investigated 26 myxoid liposarcomas (11 pure myxoid grade I, 15 myxoid/round cell grade II/III) for the hTERT expression level and compared the results of the new method with former measurements performed in silver-stained polyacrylamide gels. Both methods revealed similar results, with real-time RT-PCR being the more accurate quantification technique, which also saves time and material. Elevated hTERT expression (cut-off ratio x 100 at 1.3) was an indicator of round cell components and hence for tumor progression in myxoid liposarcoma. The new method is capable of differentiating between pure myxoid and myxoid/round cell liposarcomas for hTERT-expression more accurately.

Computer Systems↗

PubMed1987

Human immunodeficiency virus reverse transcriptase. General properties and its interactions with nucleoside triphosphate analogs.

Using affinity purified human immunodeficiency virus (HIV) reverse transcriptase the reaction assay conditions were determined. The optimum incorporation of dTMP into the (rA)n(dT)10 template with HIV reverse transcriptase required 6 mM MgCl2 and 80 mM KCl. The template specificity of HIV reverse transcriptase is quite different from those of the human gamma-polymerase-associated reverse transcriptase or avian virus reverse transcriptase. The preferential inhibition of HIV reverse transcriptase as compared to human gamma-reverse transcriptase was observed with several nucleoside analog triphosphates. The Ki values for thymidine triphosphate analogs with HIV reverse transcriptase ranged from 5 to 13 nM with decreasing effectiveness for 3'-fluoro greater than 3'-amino greater than 2',3'-dideoxy greater than 3'-azido groups. This study provides information on the structure activity relationships of the triphosphate analogs inhibitory effects on HIV reverse transcriptase versus human gamma-polymerase-associated reverse transcriptase, and the possible mechanisms of action of 3' azido thymidine and the 2',3'-dideoxynucleosides, and also identifies other nucleoside analogs for possible development as inhibitors of HIV.

Deoxyguanine Nucleotides↗

PubMed1992

RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.

High-affinity ligands of the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) were isolated by the SELEX procedure (systematic evolution of ligands by exponential enrichment) from RNA populations randomized at 32 positions. Analysis of these ligands revealed a pseudoknot consensus with primary sequence bias at some positions. We demonstrated that at least one of the ligands inhibits cDNA synthesis by HIV reverse transcriptase but fails to inhibit other reverse transcriptases. These experiments highlight the power of SELEX to yield highly specific ligands that reduce the activity of target proteins. Such ligands may provide therapeutic reagents for viral and other diseases.

Antiviral Agents↗

PubMed1992

Exogenous primer-independent cDNA synthesis with commercial reverse transcriptase preparations on plant virus RNA templates.

Upon reverse transcription and cloning manipulations with virion RNAs of several plant viruses, namely beet yellows virus, brome mosaic virus, and potato virus X, we came across a significant background synthesis of cDNA on the virion RNA template in vitro independent of exogenous primers added. When tested with beet yellow virus RNA template, several commercial preparations of avian myeloblastosis virus (AMV) reverse transcriptase showed the background activity monitored by the [alpha-32P]dNTP incorporation in vitro, while the enzyme from murine moloney leukemia virus (MMLV) was found strictly exogenous-primer-dependent. To detect possible nucleic acid contaminations in reverse transcriptase, the enzyme preparations from several commercial sources were incubated with [gamma-32P]ATP and polynucleotide kinase. The labeled material from AMV reverse transcriptase preparations comigrated with a tRNA marker in polyacrylamide gels and was found to be RNase-sensitive. The MMLV reverse transcriptase preparations were free from such a contamination. These results indicate that the exogenous-primer-independent cDNA synthesis by some AMV reverse transcriptases could be due to a contaminating tRNA (or its low-molecular-weight degradation products) serving as an endogenous primer.

Artifacts↗

PubMed1986

Biochemical heterogeneity of reverse transcriptase purified from the AIDS virus, HTLV-III.

The reverse transcriptase from AIDS virus, HTLV-III, was purified and characterized. The purified enzyme has a very high affinity for template primers (rC)n X (dG)12 and (rCm)n X (dG)12 compared to that for (rA)n X (dT)12. In addition, the HTLV-III reverse transcriptase was able to transcribe (rAm)n X (dT)12 very efficiently. The ionic requirements are unique in the sense that HTLV-III reverse transcriptase prefers Mg2+ as divalent ions to transcribe (rC)n X (dG)12 and (rA)n X (dT)12. The Mr of the enzyme is 95 000-98 000. Unlike the HTLV-I reverse transcriptase, the HTLV-III enzyme is highly stable and has a much higher activity in the presence of (rC)n X (dG)12; the Vmax for HTLV-III reverse transcriptase is several-fold higher than that for HTLV-I enzyme. The enzyme activity of the purified reverse transcriptase from HTLV-III was resolved into two peaks on a preparative isoelectric column, one at pH 5.75 and the other at pH 6.25. This leads us to conclude that the reverse transcriptase of HTLV-III is biochemically heterogeneous.

Cations, Divalent↗

PubMed1983

Mechanism of action of the endonuclease associated with the alpha beta and beta beta forms of avian RNA tumor virus reverse transcriptase.

Preparations of the alphabeta and the betabeta forms of reverse transcriptase from the Prague C strain of Rous sarcoma virus grown in chicken embryo fibroblasts, the alphabeta and the betabeta forms of the enzyme from the B77 strain of Rous sarcoma virus grown in duck embryo fibroblasts, and the alphabeta form of reverse transcriptase from avian myeloblastosis virus have been analyzed. All these enzyme preparations contain a Mn(2+) -activated endonuclease activity. The betabeta form of enzyme, in addition, contains a Mg(2+) -dependent endonuclease. Such an activity is barely detectable in the alphabeta form of enzymes. The endonuclease associated with reverse transcriptase introduces single- and double-strand breaks containing 3' OH and 5' P termini into RF I DNA. The conversion of RF I DNA to RF III DNA is more readily catalyzed by the betabeta form of reverse transcriptase. In contrast to a recently published report by Hizi et al. (J. Virol 41:974-981, 1982), we have failed to detect the conversion of RF I DNA to covalently closed relaxed circles (RF IV DNA) by any of the alphabeta form of enzymes tested. RF IV DNA was not produced by the betabeta form of reverse transcriptase either. We conclude that topoisomerization is not an intrinsic activity of reverse transcriptase. Although the conversion of RF I DNA to RF II DNA was found to be rapid, the endonuclease associated with reverse transcriptase acted slowly on RF II, RF III, and RF IV DNAs. Circular and linear single-stranded DNAs were also susceptible to cleavage by the endonuclease at a rate comparable to nicking of RF I DNA. This pattern of activity suggests that the endonuclease cleaves the RF I DNA in the single-stranded regions of the DNA induced by its supercoiling. The preference of the alphabeta and the betabeta forms of the endonuclease for viral DNA was tested with Rous-associated virus type 2 and Rous sarcoma virus transformation-defective Schmidt-Ruppin B strain DNA molecularly cloned in plasmid pBR322 and M13 DNA vectors, respectively. The rate of nicking of RF I DNA containing viral DNA or partial sequences of viral DNA with one or two tandem long terminal repeats was the same as when these sequences were not present in the host vectors. A similar lack of preference was observed with single-stranded M13 DNAs.

Avian Leukosis Virus↗

PubMed2000

Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities.

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

Amino Acid Sequence↗

PubMed1990

Interactions with tRNA(Lys) induce important structural changes in human immunodeficiency virus reverse transcriptase.

Retroviral RNA-dependent DNA polymerase (reverse transcriptase or RT) uses the 3'OH end of a cellular tRNA as primer to initiate DNA synthesis. Previous work with avian retrovirus has shown that reverse transcriptase is implicated in the selection of cellular virion-encapsidated tRNAs and has shown that the primer tRNA is positioned on the primer binding site near the 5' end of the viral RNA. These mechanisms support the idea that the retroviral polymerase should form complexes with primer tRNA and the specific encapsidated ones. The genomic sequence of human immunodeficiency virus (HIV) allows the prediction that tRNA(Lys3) is the natural primer. In this article we show, using the mobility shift assay, that recombinant HIV reverse transcriptase is able to form a complex with bovine tRNA(Lys.) By fluorescence studies and alpha-chymotrypsin analysis we have observed a modification of the enzyme conformation when reverse transcriptase is bound to the putative primer tRNA. This structural change is specific for tRNA(Lys) although the retroviral polymerase is able to interact with other tRNAs.

HIV↗

PubMed1996

Synthesis and biological evaluation of certain alkenyldiarylmethanes as anti-HIV-1 agents which act as non-nucleoside reverse transcriptase inhibitors.

Several novel alkenyldiarylmethane (ADAM) non-nucleoside HIV-1 reverse transcriptase inhibitors were synthesized. The most potent of these proved to be 3',3"-dibromo-4',4"-dimethoxy-5'5"-bis(methoxycarbonyl)-1,1-diphenyl-1-+ ++heptene (8) ADAM 8 inhibited the cytopathic effect of HIV-1 in CEM cell culture with an EC50 value of 7.1 microM and was active against an array of laboratory strains of HIV-1 in CEM-SS and MT-4 cells, but was inactive as an inhibitor of HIV-2. In common with the other known non-nucleoside reverse transcriptase inhibitors, ADAM 8 was an effective inhibitor of HIV-1 reverse transcriptase (IC50 1 microM) with poly(rC).oligo(dG), but not with poly(rA).oligo(dT), as the template/primer. ADAM 8 was inactive against HIV-1 reverse transcriptases containing non-nucleoside reverse transcriptase inhibitor resistance mutations at residues 101, 106, 108, 139, 181, 188, and 236, while it remained active against enzymes with mutations at residues 74, 98, 100, 103, and at 103/181. An AZT-resistant virus having four mutations in reverse transcriptase was more sensitive to inhibition by ADAM 8 than the wild-type HIV-1. In addition, ADAM 8 displayed synergistic activity with AZT, but lacked synergy with ddI. ADAM 8 or a structurally related analog may therefore be useful as an antiviral agent in combination with AZT or with other NNRTIs that are made ineffective by mutations at residues which do not confer resistance to ADAM 8.

Amino Acid Sequence↗

PubMed2000

The majority of duck hepatitis B virus reverse transcriptase in cells is nonencapsidated and is bound to a cytoplasmic structure.

The hepadnavirus reverse transcriptase binds cotranslationally to the viral pregenomic RNA. This ribonucleoprotein complex is then encapsidated into nascent viral core particles, where the reverse transcriptase copies the viral RNA into DNA. Here we report that 75% of the duck hepatitis B virus reverse transcriptase present in transfected LMH cells does not follow this well-known pathway but rather exists in the cell separate from the core protein or nucleocapsids. The nonencapsidated reverse transcriptase is also abundant in infected duck liver. The nonencapsidated reverse transcriptase exists as a complex set of isoforms that are most likely produced by posttranslational modification. Interestingly, only the smallest of these isoforms is encapsidated into viral core particles. The nonencapsidated reverse transcriptase is bound to a large cellular cytoplasmic structure(s) in a detergent-sensitive complex. The cellular distribution of the reverse transcriptase only partially overlaps that of the core protein, and this distribution is unaffected by blocking encapsidation. These observations raise the possibilities that the metabolic fate of the reverse transcriptase may be posttranscriptionally regulated and that the reverse transcriptase may have roles in the viral replication cycle beyond its well-known function in copying the viral genome.

Animals↗

PubMed1995

The large subunit of HIV-1 reverse transcriptase interacts with beta-actin.

HIV-1 reverse transcriptase is a dimeric enzyme mainly involved in the replication of the viral genome. A filamentous phage cDNA expression library from human lymphocytes was used to select cellular proteins interacting with HIV-1 reverse transcriptase Affinity selections using the bacterially expressed monomeric large subunit of reverse transcriptase (p66) yielded host beta-actin. This clone was expressed as glutathione-S-transferase fusion protein which was identified by using a specific antibody against beta-actin. Furthermore we show that also the eukaryotic beta-actin binds to either the large subunit of reverse transcriptase or to the Pol precursor polyprotein in vitro. The reverse transcriptase/beta-actin interaction might be important for the secretion of HIV-1 virions.

Actins↗

PubMed1995

Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR.

The secondary structure in mRNA is essential for many processes, but it can present a technical problem in making full-length cDNA with reverse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary structure, which can alter the products obtained by reverse-transcribing RNA and then PCR-amplifying the product (RT-PCR). We have been interested in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have tested the ability of several reverse transcriptases to reverse transcribe the 3'-untranslated region (3'UTR), a region that contains substantial secondary structure. When low levels of either total RNA or poly(A)+ mRNA were used, we found avian myeloblastosis virus reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-RT is inhibited by tRNA--which should make it less effective than Moloney murine leukemia virus reverse transcriptase (MMLV-RT) at reverse-transcribing total RNA--adding tRNA to poly(A)+ RNA actually increased the amount of specific RT-PCR product obtained with AMV-RT while it decreased the amount of product and enhanced mispriming with MMLV-RT. We found that pre-incubation of the oligo(dT) primer with total RNA at elevated temperature prior to reverse transcription improved the efficiency of both native and modified MMLV-RTs. These findings support the concept that secondary structures in RNA differentially affect the abilities of different reverse transcriptases to detect transcript diversity and raise the possibility that such structures could affect quantitation using RT-PCR with internal mRNA standards.

Avian Myeloblastosis Virus↗

PubMed1994

Role of RNA in enzymatic activity of the reverse transcriptase of hepatitis B viruses.

The hepadnavirus reverse transcriptase is a multifunction enzyme. In addition to its role in DNA synthesis, the polymerase is required for RNA packaging and also functions as the primer for minus-strand DNA synthesis. Previously, we demonstrated that the protein-priming activity of the polymerase requires a viral RNA segment, termed epsilon, which serves as a template for the synthesis of a short DNA oligomer that is covalently attached to the reverse transcriptase (G.-H. Wang and C. Seeger, J. Virol. 67:6507-6512, 1993). We now report that epsilon is sufficient for activation of the reverse transcriptase to prime DNA synthesis through the formation of a stable RNA-protein (RNP) complex. We also demonstrate that the binding reaction depends on sequence-specific determinants on epsilon. Moreover, our results indicate that two genetically separated domains of the reverse transcriptase are required for formation of the RNP complex. Finally, we show that the polymerase has a DNA polymerase activity in the absence of epsilon which does not depend on the protein-priming mechanism.

Animals↗

PubMed1994

Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase.

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.

Base Sequence↗

PubMed1978

Binding of tRNA to reverse transcriptase of RNA tumor viruses.

The interaction of tRNA with the reverse transcriptase (RNA-dependent DNA polymerase) of mammalian RNA viruses, such as Moloney murine leukemia virus and simian sarcoma virus, has been studied. Whereas the purified reverse transcriptase of mammalian viruses sedimented in glycerol gradients as a globular protein with a molecular weight of 70,000, after interaction with tRNA the enzyme cosedimented with a protein of 150,000 molecular weight. The twofold increase in molecular weight could be a result of either two reverse transcriptase molecules complexed with a tRNA or, alternatively, several tRNA molecules bound to a single enzyme polypeptide. The enzyme complexes were dissociated in part upon degradation of the tRNA moiety by pancreatic RNase A. The reverse transcriptase released from virions of Moloney murine leukemia virus, simian sarcoma virus, and avian myeloblastosis virus, by nonionic detergent, migrated faster on glycerol gradients than purified enzyme preparation. This phenomenon was probably due to complex formation between part of the virion enzyme and the tRNA, which is endogenous in virions. Addition of exogenous tRNA was needed, however, to quantitatively complex all the virion reverse transcriptase of Moloney murine leukemia virus and simian sarcoma viruses. The reverse transcriptase of Moloney murine leukemia virus did not show tRNA species specificity in the binding reaction when glycerol gradients were used for assay. Thus, several tRNA species of Escherichia coli, yeast, chicken, and rat origin were able to complex with the enzyme. The species specificity in the interaction between tRNA and avian myeloblastosis virus reverse transcriptase was also examined. We demonstrated that under our experimental conditions, this enzyme binds different tRNA species of E. coli and yeast as well as tRNA of chicken origin.

Animals↗

PubMed1998

Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes.

Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.

Base Sequence↗

PubMed2001

Specific detection of RT activity in culture supernantants of retrovirus-producing cells, using synthetic DNA as competitor in polymerase enhanced reverse transcriptase assay.

The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.

Animals↗

PubMed1992

Measurement of HIV-1 reverse transcriptase by a nonradioactive assay system.

Reverse transcriptase activity was measured by incorporation of dUMP linked to digoxigenin into a suitable template-primer molecule. Incorporation was monitored by using peroxidase-conjugated Fab fragments directed against digoxigenin. The standard assay measuring incorporation of radiolabeled nucleotides into acid-precipitable material was compared with this new immunochemical assay with regard to its usefulness for testing inhibitors of reverse transcriptase.

Digoxigenin↗