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PubMed2006

Reverse transcriptase template switching and false alternative transcripts.

Reverse transcriptase (RT) can switch from one template to another in a homology-dependent manner. In the study of eukaryotic transcripts, this propensity of RT can produce an artificially deleted cDNA, which can be wrongly interpreted as an alternative transcript. Here, we have investigated the presence of such template-switching artifacts in cDNA databases, by scanning a collection of human splice sites (Information for the Coordinates of Exons, ICE database). We have confirmed several cases at the experimental level. Artifacts represent a significant portion of apparently spliced sequences using noncanonical splice signals but are rare in the context of the whole database. However, care should be taken in the annotation of alternative transcripts, especially when the RT used is poorly thermostable and when the putative intron is flanked by direct repeats, which are the substrate for template switching.

Alternative Splicing↗
PubMed1996

The K65R mutation confers increased DNA polymerase processivity to HIV-1 reverse transcriptase.

The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.

DNA↗
PubMed1989

Differential inhibition of HIV-reverse transcriptase and various DNA and RNA polymerases by some catechin derivatives.

The two components of Camellia sinensis (green tea) (i.e., (-)-epigallocatechin gallate and (-)-epicatechin gallate were found to differentially inhibit the activities of reverse transcriptase and cellular DNA and RNA polymerases. The strongest inhibition by these compounds was observed with reverse transcriptase. DNA polymerases alpha and beta were also strongly inhibited. The mode of the inhibition of reverse transcriptase and other DNA polymerases was competitive with respect to the template.primer, whereas the mode the inhibition of RNA polymerase was competitive with respect to the nucleotide substrate.

Catechin↗
PubMed2006

Human normal T lymphocytes and lymphoid cell lines do express alternative splicing variants of human telomerase reverse transcriptase (hTERT) mRNA.

Alternative splicing of telomerase reverse transcriptase (hTERT) mRNA is known to contribute to regulation of telomerase activity in normal and cancerous cells, however, previous studies indicated that normal human T and B cells exhibited constitutive expression of full-length hTERT mRNA without splicing variants and that activation of telomerase upon stimulation of the cells was due to the shuttling of hTERT protein from cytoplasm to nucleus [Proc. Natl. Acad. Sci. USA 96 (1999) 5147; J. Immunol. 166 (2001) 4826]. We found that typical variants of hTERT mRNA were widespread in human lymphocyte-derived cell lines and normal stimulated T cells. In activated T cells, induction of the full-length hTERT mRNA was coupled with increased hTERT protein expression and telomerase activity. Collectively, human normal and malignant lymphocytes, like other human cells, express splicing variants of hTERT mRNA and require transcriptional activation of the hTERT gene to acquire telomerase activity.

Alternative Splicing↗
PubMed1976

Reverse transcriptase in leukocytes of leukemic patients in remission.

A cytoplasmic particulate fraction from human leukemic cells has been shown to contain reverse transcriptase and its associated high-molecular weight RHA template. We attempted to detect the reverse-transcriptase-template complex in morphologically normal peripheral blood leukocytes from patients with acute leukemia in complete remission. Our assay system consisted of a velocity glycerol gradient and cesium sulfate equilibrium gradient analysis of the endogenous reverse transcriptase reaction product. Three of nine patients in remission had positive reactions determined by glycerol gradient analysis, and eight of 10 patients in remission had positive reactions by cesium sulfate gradient analysis. We were unable to detect the template complex in leukocytes of normal persons. Thus, normal-appearing leukocytes in the peripheral blood of some leukemia patients in remission seem to retain a number of biochemical characteristics, possibly viral related, associated with leukemic cells.

Adult↗
PubMed2000

Intracellular studies of the nucleoside reverse transcriptase inhibitor active metabolites: a review.

Nucleoside reverse transcriptase inhibitors (NRTIs) plasma concentrations do not correlate with clinical efficacy or toxicity. These agents need to be phosphorylated to become active against HIV-infection. Thus, the characterization of the NRTIs intracellular metabolite pharmacological parameters will provide a better understanding that could lead to the development of more rational dose regimens in the HIV-infected population. Furthermore, intracellular measurements of NRTIs may provide a better marker with respect to clinical efficacy and toxicity than plasma concentrations. Thus, in this article we review the latest information regarding the intracellular pharmacological parameters of zidovudine (ZDV) and lamivudine (3TC) active metabolites in HIV-infected patients including the results from our recent clinical studies. We will start the discussion with ZDV and 3TC clinical efficacy, followed by systemic pharmacokinetics studies. We will then discuss the in vitro and in vivo intracellular studies with particular emphasis in the method development to measure these metabolites and we will conclude with the most current data from our clinical trials.

Adult↗
PubMed2005

Determining suitable internal standards for mRNA quantification of increasing cancer progression in human breast cells by real-time reverse transcriptase polymerase chain reaction.

Real-time reverse transcriptase polymerase chain reaction is recognized as a highly sensitive and specific method for quantification of mRNA expression. SYBR green I dye simplifies the experimental design but introduces the need for specific controls to maintain high specificity. Due to this increased sensitivity, standards that may have been acceptable for normalization of less sensitive methods have been shown to vary considerably among cell lines, tissues, proliferative states, treatments, and developmental conditions and by degree of cancer progression. It has become evident that determination of suitable normalization standards is a requirement for the use of this method as it is applied toward any new experimental model. We have assessed the suitability of a number of commonly used standards for the normalization of mRNAs among a set of human breast cancer cell lines of increasing metastatic potential and have determined that 18S rRNA and beta-actin (ACTB) mRNA are both suitable for this purpose, with each having some limitations. 18S rRNA varies less among the cell lines but has a higher degree of random variability, while ACTB mRNA varies more among cell lines but has a lower degree of random variation.

Actins↗
PubMed2004

Single unpaired nucleotides facilitate HIV-1 reverse transcriptase displacement synthesis through duplex RNA.

During reverse transcription of viral RNA, HIV-1 reverse transcriptase (RT) encounters RNA stem-loop structures that require displacement synthesis activity in which RT disrupts the RNA helix to access the template strand. A primer extension assay was developed to assess HIV-1 RT RNA displacement synthesis activity in vitro. Initial results revealed that HIV-1 RT performs only limited amounts of RNA displacement through long stretches of RNA duplex, with the majority of synthesis stalling at sequence-dependent pause positions. DNA displacement synthesis through the same sequence, however, proceeded rapidly to the end of the template. The RNA folding algorithm mfold indicated that the presence of an unpaired nucleotide, or "bulge," along the RNA duplex would promote helix melting ahead of the DNA primer terminus to create a small gap of nondisplacement synthesis. Primer extension assays using substrates possessing single-nucleotide bulges in the nontemplate strand near pause sites resulted in diminished pausing at positions within the predicted melted region. Surprisingly, the bulges also reduced pausing distal to the bulge at positions that are expected to remain base-paired. Further analysis revealed that stalling during RNA displacement synthesis results from the displaced RNA re-annealing to the template strand thus forcing the primer terminus to become unpaired and, therefore, not extendable. Introduction of a bulge facilitates displacement synthesis through distal regions by increasing RT processivity in the vicinity of a bulge and reducing the impact of branch migration on primer extension.

Base Sequence↗
PubMed1999

Detection of equine arteritis virus in semen by reverse transcriptase polymerase chain reaction-ELISA.

The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the sequence of reference strain Bucyrus, the analysed samples were 78-100% identical and showed a 84-97% nucleotide identity with Bucyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extracted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that this could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be sensitive, specific and above all directly applicable to semen. Additionally, the short time needed for the overall procedure makes this method suitable for diagnostic purposes.

Animals↗
PubMed1994

Comparison of three different crystal forms shows HIV-1 reverse transcriptase displays an internal swivel motion.

BACKGROUND: Reverse transcriptase (RT) from HIV-1 is responsible for replicating the single-stranded RNA genome to double-stranded DNA. The three-dimensional structure of RT shows that it is a strikingly asymmetric heterodimer consisting of two differently folded subunits (molecular weights 66 kDa and 51 kDa) with identical amino-terminal amino acid sequences (residues 1-428). The large active site cleft is composed of subdomains named 'finger', 'palm' and 'thumb'. There is also an RNAse H domain. RESULTS: We have compared four RT structures. The structures of two independent RT heterodimers comprising the asymmetric unit of an orthorhombic crystal form have been determined by molecular replacement and are noticeably different from each other. Comparison of the molecules in this crystal form with the two previously reported RT structures shows a related pattern of variations in relative sub domain positions. The structural differences can be described as a molecular twist between the polymerase active site located on the finger and palm domains of p66 and the rest of the molecule. This twist occurs around an axis which runs from the p66 palm domain through the p66/p51 connection domain interface and which exits below the RNAse H domain. CONCLUSIONS: From the differences in the four RT structures we infer that the molecule has a specific flexibility that allows rotation of the polymerase active site relative to the rest of the molecule. The observed swivelling motion of RT may allow the polymerase to accommodate the rotational and translational movements of the growing nucleic acid duplex, which present an especial problem for RT because it uses an asymmetric molecule (tRNA(Ly3)) as a primer for first strand synthesis.

Computer Graphics↗
PubMed2003

Colinearity of reverse transcriptase inhibitor resistance mutations detected by population-based sequencing.

High-level resistance to multiple drugs is often detected by directly sequencing uncloned polymerase chain reaction products (population-based sequencing). It is not known, however, if this method of identifying mutations gives an accurate picture of individual viral genomes. To determine how often multidrug-resistant isolates consist of clones containing every mutation present in the population-based sequence, a mean of 2.8 molecular clones was sequenced from the plasma of 25 heavily treated persons whose population-based sequence contained multiple reverse transcriptase (RT) inhibitor resistance mutations (71 clones). The 25 population-based sequences contained a mean of 5.7 nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations and 1.2 nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations. The 71 clones contained a mean of 5.3 NRTI resistance mutations and 1.0 NNRTI resistance mutations. Sequences of clones closely resembled the population-based sequence: 36 (51%) clones had each of the RT inhibitor mutations present in the population-based sequence, 25 (35%) had all but 1 RT inhibitor mutation, 4 (6%) had all but 2 RT inhibitor mutations, 3 (4%) had all but 3 RT inhibitor mutations, and 3 (4%) had all but 4 RT inhibitor mutations. Phenotypic testing of 29 clones showed that most clones were resistant to nearly all NRTIs and that those with NNRTI resistance mutations were also resistant to multiple NNRTIs. These data show that in heavily treated persons, most RT inhibitor resistance mutations are present in the same viral genomes (colinear) and that multidrug resistance often occurs within individual clones as well as within virus populations.

Amino Acid Sequence↗
PubMed1989

Inhibition of HIV-1 reverse transcriptase by 5'-triphosphates of 5-substituted uridine analogs.

The 5'-triphosphates of some 5-substituted 2'-deoxyuridine analogs were investigated for their effects on purified recombinant reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) as well as cellular DNA polymerase alpha. The triphosphates were competitive inhibitors of the viral enzyme with dTTP as the variable substrate and poly(rA)oligo(dT) as template, and preferentially inhibited the viral polymerase. Ordering the compounds according to their decreasing binding affinities, as reflected by their increasing inhibition constants for the reverse transcriptase, gave nPrearaUTP greater than nPrdUTP greater than EtdUTP greater than nPredUTP greater than HMdUTP greater than CEdUTP. Although nPredUTP was less inhibitory than nPrearaUTP under conditions of competitive inhibition, nPredUTP caused a time- and concentration-dependent inactivation of reverse transcriptase activity when preincubated with template. This inactivation was not reversed by excess dTTP. The decrease in template-primer activity did not occur with nPrearaUTP, but was shown with the chain-terminating 5'-triphosphates of 3'-fluoro- and 3'-azidothymidine. As nPredUTP, but not nPrearaUTP, was an alternative substrate, shown by the ability to support DNA synthesis in absence of competing substrate, the incorporation of nPredUTP into the primer-template apparently leads to increased inhibition of the enzyme.

Antiviral Agents↗
PubMed1992

Functional analysis of primers and templates in the synthesis of DNA catalyzed by human immunodeficiency virus type 1 reverse transcriptase.

The kinetics of copying of poly(A).(dT)n, poly(A).(U)n, poly(dA).(dT)n and poly(A).(dT)9-U by reverse transcriptase of human immunodeficiency virus-1 (HIV-1) has been studied and the binding affinity of the enzyme, for template or primer, determined. Short oligonucleotides and dTTP served as primers in the HIV-1 reverse-transcriptase-dependent DNA synthesis. Km and Vmax were measured as functions of the primer chain length; the logarithm of the values of both Km and Vmax increased linearly up to 10. For longer primers (n = 11 to n = 24) the increase of those values changes very little. The enhanced affinity of the primers, (dT)n or (U)n due to the formation of one complementary pair, A.dT, dA.dT, A.U was estimated as a factor of 2. A specific property of HIV-1 reverse transcriptase compared with other DNA polymerases (procaryotes, eucaryotes, other retroviruses and archaebacteria) was its higher affinity to riboprimers as compared to deoxyriboprimers. Relative initial rates when copying poly(A) or poly(dA) templates using different primers and various conditions were compared; the optimal temperature for the reaction of polymerization with poly(A) or poly(dA) templates and (U)10, (dT)10 or (dT)9-U primers was determined. The maximal activity of the enzyme in the case of poly(A) and decanucleotide primers was found at temperatures between 27-31 degrees C. An increase in the primer length results in the stabilization of the template.primer duplex complexed to the enzyme, thus increasing to more than 40 degrees C the optimal temperature of polymerization. The activation energy (Ea) values of the polymerization reaction for different template.primer complexes were evaluated.

DNA Replication↗
PubMed2004

Efavirenz therapy in rhesus macaques infected with a chimera of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type 1.

The specificity of nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for the RT of human immunodeficiency virus type 1 (HIV-1) has prevented the use of simian immunodeficiency virus (SIV) in the study of NNRTIs and NNRTI-based highly active antiretroviral therapy. However, a SIV-HIV-1 chimera (RT-SHIV), in which the RT from SIVmac239 was replaced with the RT-encoding region from HIV-1, is susceptible to NNRTIs and is infectious to rhesus macaques. We have evaluated the antiviral activity of efavirenz against RT-SHIV and the emergence of efavirenz-resistant mutants in vitro and in vivo. RT-SHIV was susceptible to efavirenz with a mean effective concentration of 5.9 +/- 4.5 nM, and RT-SHIV variants selected with efavirenz in cell culture displayed 600-fold-reduced susceptibility. The efavirenz-resistant mutants of RT-SHIV had mutations in RT similar to those of HIV-1 variants that were selected under similar conditions. Efavirenz monotherapy of RT-SHIV-infected macaques produced a 1.82-log-unit decrease in plasma viral-RNA levels after 1 week. The virus load rebounded within 3 weeks in one treated animal and more slowly in a second animal. Virus isolated from these two animals contained the K103N and Y188C or Y188L mutations. The RT-SHIV-rhesus macaque model may prove useful for studies of antiretroviral drug combinations that include efavirenz.

Alkynes↗
PubMed2005

Hypophosphoric acid is a unique substrate of pyrophosphorolysis catalyzed by HIV-1 reverse transcriptase.

Pyrophosphate analogues, namely, pyrophosphorous, hypophosphoric, and hypophosphorous acids, were evaluated as inhibitors in elongation reactions and substrates in pyrophosphorolysis reaction catalyzed by HIV-1 reverse transcriptase and DNA polymerase I (the Klenow fragment). The substrate efficacy of hypophosphoric acid in pyrophosphorolysis reaction exceeded that of pyrophosphate for both enzymes by more than ten times. The product of the reaction was a dNTP analogue bearing a hypophosphate in the beta,gamma-position. Pyrophosphorous and hypophosphorous acids were neither inhibitors nor substrates for the enzymes. Kinetic parameters of the pyrophosphorolysis reaction catalyzed by HIV reverse transcriptase in the presence of hypophosphoric acid were evaluated. The dTMP analogue bearing a hypophosphate in the beta,gamma-position was synthesized and its substrate properties in elongation reaction catalyzed by HIV-1 reverse transcriptase were similar to those of natural dTTP. Hypophosphoric acid was capable of removing ddTMP, ddTMP(3'N3), and ddTMP(3'NH2) from the 3'-end of primers with an equal efficacy.

Base Sequence↗
PubMed2003

Novel pharmacophore-based methods reveal gossypol as a reverse transcriptase inhibitor.

In a program to identify new structural entities for the inhibition of the HIV-1 reverse transcriptase (RT) enzyme via database searching, a series of RT pharmacophores were developed. By utilising a novel filtering technique, the National Cancer Institute database of compounds was scanned producing 15 compounds to be screened for activity. A notable inclusion was a series of gossypol derivatives. The testing of a series of compounds revealed the parent compound gossypol to be an HIV-1 reverse transcriptase inhibitor. These results suggest that at least a part of its anti-HIV activity is due to gossypol targeting the non-nucleoside inhibitor binding pocket of RT.

Databases, Factual↗
PubMedDate not supplied

Comparative study of non nucleoside inhibitors with HIV-1 reverse transcriptase based on 3D-QSAR and docking.

The intermolecular interaction between two types of non nucleoside reverse transcriptase inhibitors (NNRTIs), HEPT and TIBO, and HIV reverse transcriptase receptor (HIVRT) was investigated. The result of docking study showed that two types of NNRTIs presented similar interaction mechanism with HIVRT. The most active compound of every type of inhibitors could form one hydrogen bond with the residue Lys101 and has hydrophobic interaction with residues Tyr181, Tyr188 and Tyr318, etc. Three 3D-QSAR models including two partial correlation models (one for each family of HEPT and TIBO) and a mixed model gathering two families were constructed. Comparative study of these models indicated that the mixed model offered the strongest prediction ability. For this model, the cross-validated q2 values were 0.720 and 0.675, non-cross-validated r2 values were 0.940 and 0.920 for CoMFA and CoMSIA, respectively. It has been validated by using a test set of 27 inhibitors. Compared with previously reported works, our model showed better prediction ability. It could help us to insight the interaction between NNRTIs and HIVRT, and to design new anti-HIV NNRTIs inhibitors.

Drug Interactions↗
PubMed1991

The cauliflower mosaic virus reverse transcriptase is not produced by the mechanism of ribosomal frameshifting in Saccharomyces cerevisiae.

The capsid protein and the reverse transcriptase of cauliflower mosaic virus (CaMV) are encoded by two genes (ORF IV and ORF V) that lie in different translation reading frames. A comparison can be drawn between the synthesis of both CaMV proteins and the fusion protein in a yeast retrotransposon, Ty, resulting from a +1 frameshifting event which fuses two out-of-phase ORFs encoding the structural protein and the reverse transcriptase of Ty. For this reason, we constructed a yeast expression vector containing CaMV ORF VII fused to CaMV ORF III by a fragment of 452 bp including the overlapping region of ORF IV and ORF V, ORF VII and ORF III being used as reporter genes. We characterized two proteins (22 and 50 kDa) synthesized from this plasmid in the yeast expression system. We demonstrated that the 50-kDa polypeptide is not synthesized from a +1 frameshifting event but is probably a dimeric form of the 22-kDa protein. From this result we conclude that the CaMV reverse transcriptase is not produced by a mechanism of ribosomal frameshifting.

Brassica↗