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PubMed1993

The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase.

A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.

Base Sequence↗

PubMed1992

Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates.

A comparison of the fidelity of reverse transcriptases (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) is made using RNA and DNA primer-template molecules in vitro. Selected template target sites containing either uracil or thymine are used to measure nucleotide insertion fidelities and to compare the efficiency of extending mismatched nucleotides at primer 3'-termini. HIV-1 reverse transcriptase is observed to incorporate as many as three consecutive mismatches and to continue efficient elongation from mismatched primer 3'-termini without discernible pausing. Nucleotide misinsertion and mispair extension efficiencies are similar for both enzymes on RNA and DNA templates having identical surrounding sequence. HIV-1 and AMV reverse transcriptases form G.T and G.U mismatches most efficiently, between 1.6 x 10(-4) and 7 x 10(-4), and both enzymes extend G.U with exceptionally high efficiencies, 2.7 x 10(-2) for HIV-1 RT and 4.5 x 10(-2) for AMV RT. Extension of the G.T mismatch is similar for AMV RT (5.8 x 10(-2) but 20-fold less efficient for HIV-1 RT. C.U and C.T mismatches are formed by both enzymes in a frequency range of 4.4 x 10(-5)-2.4 x 10(-4). HIV-1 RT extends these mismatches with slightly higher efficiencies (5.5 x 10(-3)-5.9 x 10(-3)) than AMV RT (5.6 x 10(-4)-2.1 x 10(-3)). Insertion of dTMP opposite U and T occur at about 1 x 10(-4)-2 x 10(-4) for HIV-1 RT. For AMV RT, formation of T.U mispairs occurs with an 8-fold lower efficiency, whereas insertion of dTMP opposite T is not detected. This particular DNA template sequence generates a pause site for AMV RT but not HIV-1 RT. HIV-1 RT dissociation rate constants are about 8-fold larger from a DNA primer bound to a DNA template (0.5 s-1), as compared with an RNA template (0.06 s-1) at one site, and are at most 2-fold larger at another site. The equilibrium binding constant for HIV-1 RT bound to DNA primed RNA and DNA templates appears to be similar, KD approximately 2.5 nM. Values of kpol from 0.3 to 1.5 nucleotides/s are obtained for HIV-1 RT at the RNA and DNA template sites used to measure insertion and extension fidelity. The relatively high efficiency of mispair extension catalyzed by reverse transcriptases with both RNA and DNA templates suggests that a significant component of retroviral genetic variability may be related to the ability of reverse transcriptases to continue efficient synthesis of DNA containing mismatches on both RNA and DNA templates.

Autoradiography↗

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↗

PubMed2005

Impaired rescue of chain-terminated DNA synthesis associated with the L74V mutation in human immunodeficiency virus type 1 reverse transcriptase.

The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.

Adenosine Triphosphate↗

PubMed1998

Control of initiation of viral plus strand DNA synthesis by HIV reverse transcriptase.

Human immunodeficiency virus reverse transcribes its single-stranded RNA genome making a DNA copy. As synthesis proceeds, the RNA is simultaneously degraded to oligomers; one of these, the polypurine tract, primes synthesis of a plus strand DNA. The viral reverse transcriptase (RT) degrades all of the non-polypurine tract oligomers. We show that unlike other DNA polymerases the retroviral RT can bind either end of an annealed RNA primer, the 5'-end for degradation and the 3'-end for synthesis. The competition between the two binding modes at any primer determines whether it will be extended or degraded. The 5'-end binding can be suppressed in at least two ways. The sequence of the primer can be such that a region at the 5'-end is unannealed or a DNA primer can be annealed just adjacent to the 5'-end of the RNA primer. This promotes binding of RT to the RNA 3'-end, allowing a primer that would normally be degraded to be extended. Implications for human immunodeficiency virus replication and antiviral therapy are discussed.

Binding Sites↗

PubMed1993

Isolation and molecular phylogenetic analysis of actin-coding regions from Emiliania huxleyi, a Prymnesiophyte alga, by reverse transcriptase and PCR methods.

Reverse transcriptase and polymerase chain reaction methods were used to amplify and clone actin cDNAs from the chlorophylls a + C-containing unicellular alga, Emiliania huxleyi (Prymnesiophyta). Actins in E. huxleyi are defined by a gene family containing at least six distinct coding regions that were derived from relatively recent gene duplications. Five of the coding regions (types 1, 2, and 4-6) varied only among synonymous codons. A nonsynonomous change in a sixth coding region (type 3 actin) produced a serine-to-phenylalanine replacement. The G + C composition of third positions in E. huxleyi actin genes is 98%, which contrasts with the mean value of 50% G + C content for first and second positions. Distance-matrix and parsimony analyses of actin genes identified the prymnesiophytes as a photosynthetic lineage that is not already related to other eukaryotic algal groups.

Actins↗

PubMed2004

Immortalization of human dental papilla, dental pulp, periodontal ligament cells and gingival fibroblasts by telomerase reverse transcriptase.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is catalytic subunit of human telomerase. METHODS: We studied the immortalization of a series of human dental and periodontal cells by ectopic expression of hTERT and co-expression of hTERT with human papilloma virus 16 (HPV16) or simian virus 40 (SV40). Differentiation abilities of the established cell lines were studied in terms of the mineralized matrix formation and gene expression. RESULTS: We established immortalized gingival fibroblasts by hTERT, dental papilla and periodontal ligament cells by hTERT and HPV16, and pulp cells by hTERT and SV40. The papilla and pulp cells showed mineralization and dentin sialophosphoprotein (DSPP) expression when cultured in the presence of beta-glycerophosphate. The immortalized periodontal ligament cells did not show mineralization or DSPP expression, although expressions of alkaline phosphatase, osteopontin and osteocalcin were detected. CONCLUSIONS: These cell lines will be useful tools for studying the repair and regeneration of dental and periodontal tissues and various diseases including odontogenic tumors.

Animals↗

PubMed1995

Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR.

A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV.

Animals↗

PubMed2005

Regulation of telomere length by an N-terminal region of the yeast telomerase reverse transcriptase.

Telomerase is a reverse transcriptase that maintains chromosome integrity through synthesis of repetitive telomeric sequences on the ends of eukaryotic chromosomes. In the yeast Saccharomyces cerevisiae, telomere length homeostasis is achieved through negative regulation of telomerase access to the chromosome terminus by telomere-bound Rap1 protein and its binding partners, Rif1p and Rif2p, and positive regulation by factors such as Ku70/80, Tel1p, and Cdc13p. Here we report the identification of mutations within an N-terminal region (region I) of the yeast telomerase catalytic subunit (Est2p) that cause telomere lengthening without altering measurable catalytic properties of the enzyme in vitro. These telomerase mutations affect telomere length through a Ku-independent mechanism and do not alter chromosome end structure. While Tel1p is required for expression of the telomere-lengthening phenotype, Rif1p and Rif2p are not, suggesting that telomere overextension is independent of Rap1p. Taken together, these data suggest that specific amino acids within region I of the catalytic subunit of yeast telomerase play a previously unanticipated role in the response to Tel1p regulation at the telomere.

Alleles↗

PubMed2002

Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells. It may be used as a tool for driving a gene to kill tumors specifically. To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells. A tumor-specific expression plasmid phTERT-CD was constructed, in which the E. coli cytosine deaminase (CD) gene was controlled by the hTERT promoter. A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid. It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold. Mixed cell experiments showed a strong "bystander effect" on CD-negative cells. Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c. inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days. These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer.

Animals↗

PubMed2004

Enhanced long-term survival, but no increase in replicative capacity, following retroviral transduction of human cord blood CD34+ cells with human telomerase reverse transcriptase.

Overexpression of telomerase reverse transcriptase (hTERT) can immortalize some primary human mesenchymal cells. We investigated whether retrovirally-mediated expression of hTERT in CD34+ umbilical cord blood (UCB) cells can extend the replicative lifespan of human hematopoietic progenitor cells. Overexpression of hTERT did not immortalize these cells but did lead to enhanced survival of mature hematopoietic cells.

Antigens, CD34↗

PubMed2006

Cancer immunotherapy targeting the telomerase reverse transcriptase.

The human telomerase reverse transcriptase (hTERT) is expressed in more than 85% of tumor cells but is usually not found in normal cells, which makes hTERT as an ideal tumor-associate antigen (TAA) to develop potential vaccine specifically destroying cancers without impairing normal tissues in human cancer immunotherapy. Here are reviewed the fundamental advances of studies on immunogenicity of hTERT or its peptides and the early clinical trials using the hTERT vaccine approach in the last decades.

Antigens, Neoplasm↗

PubMed2003

Human immunodeficiency virus reverse transcriptase and protease sequence database.

The HIV reverse transcriptase and protease sequence database is an on-line relational database that catalogues evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of antiretroviral therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions to GenBank, sequences published in journal articles and sequences of HIV isolates from persons participating in clinical trials. Sequences are linked to data about the source of the sequence, the antiretroviral drug treatment history of the person from whom the sequence was obtained and the results of in vitro drug susceptibility testing. Sequence data on two new molecular targets of HIV drug therapy--gp41 (cell fusion) and integrase--will be added to the database in 2003.

Amino Acid Sequence↗

PubMed2004

Topoisomerase I dissociates human immunodeficiency virus type 1 reverse transcriptase from genomic RNAs.

Both HIV-1 reverse transcriptase (RT) and topoisomerase I bind to structural RNAs and they cooperate to synthesize cDNA during the replication of HIV-1. In this study, we find that human topoisomerase I exclusively dissociated HIV-1 reverse transcriptase, which strongly binds to structural RNAs. Meanwhile, topoisomerase I did not dissociate either HIV-1 nucleocapsid proteins or murine leukemia virus RT which was bound to RNA. We propose that human topoisomerase I may regulate the binding of RT to RNAs and play a pivotal role in HIV-1 replication.

Base Sequence↗

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↗

PubMed1997

Effects of cation substitutions on reverse transcriptase and on human immunodeficiency virus production.

Reverse transcription is a key aspect of the retroviral life cycle. The enzyme reverse transcriptase requires divalent cations, manganese or magnesium, for function. In some cation-dependent systems substitution of a physiological metal by a nonphysiological metal has been shown to work. We investigated the effect of different cations on HIV reverse transcriptase activity. The studies established reaction conditions for assaying different cations. A variety of transition metals were used in in vitro assays with HIV recombinant RT homodimer and some were delivered to HIV-infected cells in vitro to study effects on virus production. Most metals substituted adequately for magnesium. However, palladium showed a marked nonreversible inhibition of RT activity in vitro that correlated with reduced HIV virus production in tissue culture. A more extensive range of transition metals and divalent cations was tested for their effects on detection of HIV RT from infected cell supernatants. In these complex phenotypes were seen. In some cases the RT activity appeared to be more easily detectable. This may relate to calcium-dependent nucleases in cell supernatants being inhibited, leading to an apparent enhancement of RT activity, or may be due to direct effects on RT processivity.

Cations↗

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↗