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PubMed2003

Mitochondrial DNA depletion and morphologic changes in adipocytes associated with nucleoside reverse transcriptase inhibitor therapy.

BACKGROUND: Nucleoside analogue reverse transcriptase inhibitor (NRTI) therapy provides sufficient conditions for progressive subcutaneous fat wasting in HIV-infected patients. As NRTI-induced host toxicity is proposed to involve cellular mitochondrial DNA (mtDNA) depletion, determinants of cellular mtDNA copy number and mitochondrial mass in adipocyte samples from NRTI-treated HIV-infected patients and antiretroviral-naive controls were investigated. Adipose tissue morphology was also assessed. METHODS: Subcutaneous fat samples were obtained from NRTI-treated, HIV-infected patients (n = 21), antiretroviral therapy-naive HIV-infected controls (n = 11), and HIV-seronegative controls (n = 6). Non-adipocytes were removed by collagenase digestion. Adipocyte mtDNA copies/cell was measured using a real time PCR-based assay, and adipocyte mitochondrial protein content was also measured. Light and electron microscopy were performed on tissue samples. FINDINGS: Adipocyte mtDNA copies/cell values were similar (P = 0.56) in HIV seronegative and HIV-infected control groups. NRTI treatment was associated with reduced adipocyte mtDNA copies/cell, representing mean mtDNA depletion in NRTI-treated individuals of 77.7% compared with the mean value for the HIV-infected control group (P < 0001). Additionally, significant differences were found in adipocyte mtDNA copies/cell between patients receiving stavudine (n = 12, mean mtDNA depletion 87.1%) and zidovudine (n = 9, mean mtDNA depletion 52.1%) (P < 0.001). Adipocyte mitochondrial mass was increased in the stavudine group only (mean increase 289%, P < 0.01). INTERPRETATION: NRTI therapy is associated with mtDNA depletion and mitochondrial proliferation in adipocytes, consistent with the hypothesis that NRTI-induced mtDNA depletion contributes to the pathogenesis of subcutaneous fat wasting. Morphologic assessment also supports a role for NRTI therapy in inducing adipocyte metabolic dysfunction and cell death.

Adipocytes↗

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↗

PubMed2000

Human telomerase activation requires two independent interactions between telomerase RNA and telomerase reverse transcriptase.

Inhibition or activation of the reverse transcriptase telomerase can profoundly affect the proliferative capacity of normal cells and cancers. Here, we elucidate structural requirements for function of the essential RNA component of human telomerase, hTR. Two motifs within the independently stable H/ACA domain of hTR are required for accumulation of the mature RNA in vivo. However, these motifs can be substituted by a heterologous H/ACA family RNA. Two additional hTR elements are required both in vivo and in vitro for telomerase catalytic activity. Surprisingly, each of these elements independently binds to the telomerase reverse transcriptase. Our results establish fundamental differences between vertebrate and ciliate telomerase ribonucleoprotein architectures and also suggest strategies for the pharmaceutical development of telomerase-based anticancer therapies.

Base Sequence↗

PubMed1996

Wheat retrotransposon families identified by reverse transcriptase domain analysis.

Variations in the reverse transcriptase domain were analyzed to clarify the family structure of retrotransposons in the wheat genome. Seven families were identified on the basis of the nucleotide sequence similarities of the domain. These families are common to the genomes of ancestral diploid species of wheat, evidence that they were established prior to diploid speciation, and their presence in barley and rice was suggested. Recent activity of some of the families was inferred in wheat on the basis of data that included synonymous and nonsynonymous nucleotide substitution rates in the reverse transcriptase domain.

Cloning, Molecular↗

PubMed2000

Identification of functionally important domains in the N-terminal region of telomerase reverse transcriptase.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase-like motifs that presumably mediate catalysis. These motifs are located in the C-terminal region of the polypeptide. Hidden Markov model-based sequence analysis revealed in the N-terminal region of all TERTs the presence of four conserved motifs, named GQ, CP, QFP, and T. Point mutation analysis of conserved residues confirmed the functional importance of the GQ motif. In addition, the distinct phenotypes of the GQ mutants suggest that this motif may play at least two distinct functions in telomere maintenance. Deletion analysis indicates that even the most N-terminal nonconserved region of yeast TERT (N region) is required for telomerase function. This N region exhibits a nonspecific nucleic acid binding activity that probably reflects an important physiologic function. Expression studies of various portions of the yeast TERT in Escherichia coli suggest that the N region and the GQ motif together may constitute a stable domain. We propose that all TERTs may have a bipartite organization, with an N-GQ domain connected to the other motifs through a flexible linker.

Amino Acid Sequence↗

PubMed2004

The crystal structure of the monomeric reverse transcriptase from Moloney murine leukemia virus.

Reverse transcriptases (RTs) are multidomain enzymes of variable architecture that couple both RNA- and DNA-directed DNA polymerase activities with an RNase H activity specific for an RNA:DNA hybrid in order to replicate the single-stranded RNA genome of the retrovirus. Previous structural work has been reported for the heterodimeric HIV-1 and HIV-2 RTs. We now report the first crystal structure of the full-length Moloney murine leukemia virus (MMLV) RT at 3.0 A resolution. The structure reveals a clamp-shaped molecule resulting from the relative positions of the thumb, connection, and RNase H domains that is strikingly different from the HIV-1 RT and provides the first example of a monomeric reverse transcriptase. A comparative analysis with related DNA polymerases suggests a unique trajectory for the template-primer exiting the polymerase active site and provides insights regarding processive DNA synthesis by MMLV RT.

Amino Acid Sequence↗

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↗

PubMed2004

Recent advances in the development of next generation non-nucleoside reverse transcriptase inhibitors.

Since their discovery, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become one of the cornerstones of highly active anti-retroviral therapy (HAART). Currently, three NNRTI agents, efavirenz, nevirapine and delavirdine are commercially available. Efavirenz and nevirapine, used in combination with nucleoside reverse transcriptase inhibitors (NRTIs), provide durable regimens with efficacy comparable to protease inhibitor (PI) containing therapies. When virological failure occurs following treatment with an NNRTI, the resistance mutations can confer reduced sensitivity to the entire agent class. Therefore, the strategy for the development of next generation NNRTIs has been to focus on compounds which have improved potencies against the clinically relevant viral mutants. Agents with improved virological profiles and which maintain the ease of administration and favorable safety profiles of the current agents should find use in anti-retroviral naïve patients as well as in components of salvage regimens in the anti-retroviral experienced patient. This review summarizes the recent developments with compounds in clinical trials as of January 2002 as well as to summarize information on new agents appearing in the primary and patent literature between January 2001 and December 2002.

Anti-HIV Agents↗

PubMed1998

The role of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy of HIV-1 infection.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have, in addition to the nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), gained a definitive place in the treatment of HIV-1 infections. Starting from the HEPT and TIBO derivatives, more than 30 structurally different classes of compounds have been identified as NNRTIs, that is compounds that are specifically inhibitory to HIV-1 replication and targeted at the HIV-1 reverse transcriptase (RT). Two NNRTIs (nevirapine and delavirdine) have been formally licensed for clinical use and several others are in preclinical or clinical development [thiocarboxanilide UC-781, HEPT derivative MKC-442, quinoxaline HBY 097 and DMP 266 (efavirenz)]. The NNRTIs interact with a specific 'pocket' site of HIV-1 RT that is closely associated with, but distinct from, the NRTI binding site. NNRTIs are notorious for rapidly eliciting resistance due to mutations of the amino acids surrounding the NNRTI-binding site. However, the emergence of resistant HIV strains can be circumvented if the NNRTIs, alone or in combination, are used from the start at sufficiently high concentrations. In vitro, this procedure has proved to 'knock-out' virus replication and to prevent resistance from arising. In vivo, various triple-drug combinations of NNRTIs (nevirapine, delavirdine or efavirenz) with NRTIs (AZT, 3TC, ddI or d4T) and/or PIs (indinavir or nelfinavir) have been shown to afford a durable anti-HIV activity, as reflected by both a decrease in plasma HIV-1 RNA levels and increased CD4 T-lymphocyte counts.

Alkynes↗

PubMedDate not supplied

Perspectives of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy of HIV-1 infection.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have, in addition to the nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs), gained a definitive place in the treatment of HIV-1 infections. Starting from the HEPT and TIBO derivatives, more than thirty structurally different classes of compounds have been identified as NNRTIs, that is compounds that are specifically inhibitory to HIV-1 replication and targeted at the HIV-1 reverse transcriptase (RT). Two NNRTIs (nevirapine and delavirdine) have been formally licensed for clinical use and several others are (or have been) in preclinical and/or clinical development [tivirapine (TIBO R-86183), loviride (alpha-APA R89439), thiocarboxanilide UC-781, HEPT derivative MKC-442, quinoxaline HBY 097, DMP 266 (efavirenz), PETT derivatives (trovirdine, PETT-4, PETT-5) and the dichlorophenylthio(pyridyl)imidazole derivative S-1153]. The NNRTIs interact with a specific 'pocket' site of HIV-1 RT that is closely associated with, but distinct from, the NRTI binding site. NNRTIs are notorious for rapidly eliciting resistance due to mutations of the amino acids surrounding the NNRTI-binding site. However, the emergence of resistant HIV strains can be circumvented if the NNRTIs, preferably in combination with other anti-HIV agents, are used from the start at sufficiently high concentrations. In vitro, this procedure has been shown to 'knock-out' virus replication and to prevent resistance from arising. In vivo, various triple-drug combinations containing NNRTIs, NRTIs and/or PIs may result in an effective viral suppression and ensuing immune recovery. However, this so-called HAART (highly active antiretroviral therapy) may also fail, and this necessitates the design of new and more effective drugs and drug cocktails.

HIV Infections↗

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↗

PubMed1993

Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase.

An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.

Biotin↗

PubMed2005

Nucleotide modification at the gamma-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase.

The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their gamma-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the gamma-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.

Binding Sites↗

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↗

PubMed2004

End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon.

The reverse transcriptase encoded by the non-long terminal repeat retrotransposon R2 has been shown to be able to jump from the 5'-end of one RNA template (the donor) to the 3'-end of a second RNA template (the acceptor) in the absence of preexisting sequence identity between the two templates. These jumps between RNA templates have similarity to the end-to-end template jumps described for the RNA-directed RNA polymerases encoded by certain RNA viruses. Here we describe for the first time the mechanism by which such end-to-end template jumps can occur. Most template jumps by the R2 reverse transcriptase are brought about by the enzyme's ability to add nontemplated (overhanging) nucleotides to the cDNA when it reaches the end of the donor RNA. The enzyme then anneals these overhanging nucleotides to sequences at the 3'-end of the acceptor RNA. The annealing is most efficient if it involves the terminal nucleotide(s) of the acceptor RNA but can occur to sites at least 5 nucleotides from the 3'-end. These end-to-end jumps are similar to steps proposed to be part of the integration reaction of non-long terminal repeat retrotransposons and can explain chimeric integration products derived from multiple RNA templates.

Cross-Linking Reagents↗