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PubMedDate not supplied

Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Animals↗
PubMed1994

Synthesis of a series of 4-(arylethynyl)-6-chloro-4-cyclopropyl-3,4-dihydroquinazolin-2(1H)-ones as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

As part of an ongoing effort to prepare novel non-nucleoside inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT), a series of 4-(arylethynyl)-6-chloro-4-cyclopropyl-3,4-dihydroquinazolin -2(1H)-ones 4aa-l has been prepared. Target compounds 4a-e were synthesized via addition of various 1-lithio-2-(aryl)alkyne nucleophiles to a 1-protected-4-cyclopropylquinazolin-2(1H)-one (7), followed by deprotection. The 3-methyl compound 4aa was prepared in an analogous manner, with the 3-alkylation performed prior to deprotection. Alternatively, the target compounds 4f-l were prepared by addition of 1-lithio-2-(trimethylsilyl)acetylene to 7, followed by deprotection and subsequent palladium-catalyzed coupling with various aryl halides. By incorporating an aryl group onto the end of the 4-acetylene functionality, the requirement for a metabolically labile 3-methyl group on the dihydroquinazolinone nucleus has been eliminated. A number of the target compounds were shown to be potent inhibitors of HIV-1 RT. Compound 4a, which had exhibited the most favorable overall biological profile, was resolved via a four-step procedure to provide the enantiomers 13a and 13b. Compound 13a having the (-)-4(S) configuration was shown to be the active enantiomer and was selected as a candidate for further investigation.

Cells, Cultured↗
PubMed2002

Cloning and characterization of rice (Oryza sativa L) telomerase reverse transcriptase, which reveals complex splicing patterns.

Plant chromosomes terminate in telomeres as in other eukaryotes. Telomeres are vital to genome stability and their malfunctioning is lethal. One of the core components of the telomere complex is telomerase. The enzyme activity depends on RNA (TER) and reverse transcriptase (TERT) subunits. We describe here the isolation, sequencing and characterization of the telomerase reverse transcriptase catalytic subunit from the monocot plant Oryza sativa L. (OsTERT). A single copy of this gene is present in the rice genome. The protein predicted from the OsTERT sequence has all the signature motifs of the TERT family members. Our data indicate that rice telomerase activity is developmentally regulated and is high in in vitro tissue and cell culture. However, steady-state transcript levels of the TERT gene do not seem to correlate with enzyme activity. Northern and RT-PCR analyses of the OsTERT gene transcript profile show multiple differentially spliced transcripts in both telomerase-positive and telomerase-negative tissues. Based on quantitative analysis of these transcripts, we speculate that the overall balance between the quantities of particular alternatively spliced transcripts may determine whether the TERT protein(s) is active or not. The diversity of splicing variants detected suggests that, as recently discovered for mammalian TERT proteins, rice TERT protein variants may perform functions other than telomere maintenance.

Alternative Splicing↗
PubMedDate not supplied

Sequence homology between retroviral reverse transcriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus.

In infected cells, the RNA genomes of RNA tumour viruses are copied into DNA by a virus-encoded reverse transcriptase enzyme. This transfer of information from RNA into DNA was thought to be a unique feature of RNA tumour viruses, but recent results suggest it may be a more general strategy. Hepatitis B virus (HBV) has a double-stranded DNA genome, and it has recently been shown that the minus DNA strand of the HBV genome is copied from a plus-strand RNA template, leading to the suggestion that reverse transcription is central to the life cycle of HBV. More recently it has been suggested that the replication cycle of a plant virus, cauliflower mosaic virus (CaMV), includes a reverse transcription step. We report here the existence of amino acid sequence homology between retroviral reverse transcriptase and the putative polymerases of HBV and CaMV.

Amino Acid Sequence↗
PubMed2003

Sensitive and specific detection of circulating cancer cells in patients with hepatocellular carcinoma; detection of human telomerase reverse transcriptase messenger RNA after immunomagnetic separation.

PURPOSE: We evaluated whether detection of human telomerase reverse transcriptase (hTERT) mRNA after immunomagnetic separation is useful to detect circulating cancer (CC) cells. EXPERIMENTAL DESIGN: Two ml of peripheral blood were collected from 55 cases with hepatocellular carcinoma (HCC), 20 cases with chronic liver diseases devoid of cancer, and 20 healthy volunteers. Then 1500 and 500 micro l were subjected to immunomagnetic separations using Ber-EP4 and anti-CD45 antibodies, harvested and supernatant cells were collected as epithelial and nonleukocyte fractions, respectively. Samples of each fraction were subjected to reverse transcription-PCR detecting beta-actin, interleukin-2 receptor (IL-2r), alpha-fetoprotein, and hTERT mRNAs. The cases were judged to be positive, equivocal, or negative for CC cells when hTERT positivity with IL-2r negativity, hTERT positivity with IL-2r positivity, or hTERT negativity was seen in epithelial and/or nonleukocyte fractions, respectively. RESULTS: The dilution experiments revealed that our system could detect 10(0-1) HeLa cells involved in 2 ml of blood. The Ber-EP4-harvested cells from cases with distant metastasis were positive for immunostaining using Hep Par 1 monoclonal antibody. CC cells were judged to be positive in 29 of 55 (53%) HCC cases. On the contrary, no cases without HCC were determined to be positive. The frequency of positivity was significantly correlated with disease extent of HCC. CONCLUSIONS: These results strongly suggest that detection of hTERT mRNA after immunomagnetic separation is a specific and sensitive tool to detect CC cells and that it would provide useful source for further investigation of cancer metastasis.

Aged↗
PubMed2000

Genetic diversity of protease and reverse transcriptase sequences in non-subtype-B human immunodeficiency virus type 1 strains: evidence of many minor drug resistance mutations in treatment-naive patients.

Most human immunodeficiency virus (HIV) drug susceptibility studies have involved subtype B strains. Little information on the impact of viral diversity on natural susceptibility to antiretroviral drugs has been reported. However, the prevalence of non-subtype-B (non-B) HIV type 1 (HIV-1) strains continues to increase in industrialized countries, and antiretroviral treatments have recently become available in certain developing countries where non-B subtypes predominate. We sequenced the protease and reverse transcriptase (RT) genes of 142 HIV-1 isolates from antiretroviral-naive patients: 4 belonged to group O and 138 belonged to group M (9 subtype A, 13 subtype B, 2 subtype C, 5 subtype D, 2 subtype F1, 9 subtype F2, 4 subtype G, 5 subtype J, 2 subtype K, 3 subtype CRF01-AE, 67 subtype CRF02-AG, and 17 unclassified isolates). No major mutations associated with resistance to nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors were detected. Major mutations linked to resistance to non-NRTI agents were detected in all group O isolates (A98G and Y181C) and in one subtype J virus (V108I). In contrast, many accessory mutations were found, especially in the protease gene. Only 5.6% of the 142 strains, all belonging to subtype B or D, had no mutations in the protease gene. Sixty percent had one mutation, 22.5% had two mutations, 9.8% had three mutations, and 2.1% (all group O strains) had four mutations. In order of decreasing frequency, the following mutations were identified in the protease gene: M36I (86.6%), L10I/V (26%), L63P (12.6%), K20M/R (11.2%), V77I (5.6%), A71V (2.8%), L33F (0.7%), and M46I (0.7%). R211K, an accessory mutation associated with NRTI resistance, was also observed in 43.6% of the samples. Phenotypic and clinical studies are now required to determine whether multidrug-resistant viruses emerge more rapidly during antiretroviral therapy when minor resistance-conferring mutations are present before treatment initiation.

Amino Acid Sequence↗
PubMed1996

Determination of HIV-1 susceptibility to reverse transcriptase (RT) inhibitors by a quantitative cell-free RT assay.

BACKGROUND: Mutations in the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) gene confer resistance to antiviral drugs acting on RT. Current methods employed to detect such resistance require time-consuming culture techniques during which selective pressures may affect the outcome of the test. OBJECTIVES: We sought to determine whether drug-susceptible and drug-resistant HIV-1 derived from clinical specimens could be distinguished by the effects of the active form of the drug on the enzyme activity in a quantitative, cell-free RT assay. STUDY DESIGN: Polyethylene glycol (PEG)-precipitated virus was obtained from 7-day culture supernatants. RT activity in the lysed viral extracts was measured in the presence of increasing concentrations of the active form of the drug being tested. IC50 (50% inhibitory concentration) values were determined by application of the median effect equation. RESULTS: Assays from nine post-nevirapine therapy isolates gave IC50 values at least 2 logs greater than pre-nevirapine isolates. The method also correctly distinguished between isolates sensitive and resistant to 2',3'-dideoxyinosine (ddI), but not between the ZDV-sensitive and ZDV-resistant isolates tested. The results agreed with data obtained by sequencing and by culture-based susceptibility assays.

Cell-Free System↗
PubMed1991

Ferrate oxidation of murine leukemia virus reverse transcriptase: identification of the template-primer binding domain.

Treatment of murine leukemia virus reverse transcriptase (MuLV RT) with potassium ferrate, an oxidizing agent known to oxidize amino acids involved in phosphate binding domains of proteins, results in the irreversible inactivation of both the DNA polymerase and the RNase H activities. Significant protection from ferrate-mediated inactivation is observed in the presence of template-primer but not in the presence of substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme loses template-primer binding activity as judged by UV-mediated cross-linking of radiolabeled DNA. Comparative tryptic peptide mapping by reverse-phase HPLC of native and ferrate-oxidized enzyme indicated the presence of two new peptides eluting at 38 and 57 min and a significant loss of a peptide eluting at 74 min. Purification, amino acid composition, and sequencing of these affected peptides revealed that they correspond to amino acid residues 285-295, 630-640, and 586-599, respectively, in the primary amino acid sequence of MuLV RT. These results indicate that the domains constituted by the above peptides are important for the template-primer binding function in MuLV RT. Peptide I is located in the polymerase domain whereas peptides II and III are located in the RNase H domain. Amino acid sequence analysis of peptides I and II suggested Lys-285 and Cys-635 as the probable sites of ferrate action.

Amino Acid Sequence↗
PubMedDate not supplied

[Cloning and expression in Escherichia coli of reverse transcriptase coded by the mobile genetic element jockey].

The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. Current models of the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As it is demonstrated here, a 2.23 kb DNA fragment from the region of the jockey encoding the putative reverse transcriptase, was stably introduced into the expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA-directed and DNA-directed DNA polymerase activities, but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulfhydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and "activated" DNA is not effective. The results of this work suggest that the RNA-directed DNA polymerase coded by jockey elements may be involved in the transcription of the elements.

Amino Acid Sequence↗
PubMedDate not supplied

Transcription of 2'-deoxy-2'-fluoro-modified and phosphorothioate-modified RNA templates by HIV-1 reverse transcriptase.

RNA templates yield the corresponding DNA in the presence of human immunodeficiency virus reverse transcriptase (HIV-1 RT). The purpose of this study was to determine whether RNA that was modified with either 2'-deoxy-2'-fluoro analogs or with internucleotide phosphorothioate linkages could serve as templates for HIV-1 RT. Modified RNA that contained either 2'-deoxy 2'-fluoro pyrimidine nucleoside analogs or internucleotide phosphorothioate diester linkages 5'- to pyrimidine nucleosides were enzymatically synthesized and tested for template activity with recombinant HIV-1 RT. RNA that was modified with either 2'-deoxy-2'-fluorouridine or with internucleotide phosphorothioate linkages 5'- to pyrimidines yielded full length HIV-1 reverse transcription products, with complete fidelity in transcription. RNA that was modified with 2'-deoxy-2'-fluorocytidine, either alone or in combination with 2'-deoxy-2'-fluorouridine, did not function as templates for HIV-1 RT, under the conditions reported here. The ability of 2'-deoxy-2'-fluoro-modified and phosphorothioate-modified RNA to serve as template for the RNA-dependent DNA polymerase of HIV-1 RT has not hitherto been reported.

Base Sequence↗
PubMed2006

The structure and function of telomerase reverse transcriptase.

The structure and integrity of telomeres are essential for genome stability. Telomere dysregulation can lead to cell death, cell senescence, or abnormal cell proliferation. The maintenance of telomere repeats in most eukaryotic organisms requires telomerase, which consists of a reverse transcriptase (RT) and an RNA template that dictates the synthesis of the G-rich strand of telomere terminal repeats. Structurally, telomerase reverse transcriptase (TERT) contains unique and variable N- and C-terminal extensions that flank a central RT-like domain. The enzymology of telomerase includes features that are both similar to and distinct from those characteristic of other RTs. Two distinguishing features of TERT are its stable association with the telomerase RNA and its ability to repetitively reverse transcribe the template segment of RNA. Here we discuss TERT structure and function; its regulation by RNA-DNA, TERT-DNA, TERT-RNA, TERT-TERT interactions, and TERT-associated proteins; and the relationship between telomerase enzymology and telomere maintenance.

Animals↗
PubMed2001

Synthesis and biological activities of potential metabolites of the non-nucleoside reverse transcriptase inhibitor efavirenz.

Studies on the biotransformation of the clinically important non-nucleoside reverse transcriptase inhibitor efavirenz have shown that oxidation and secondary conjugation are important components of the processing of this molecule in vivo. We have synthesized metabolites of efavirenz to confirm their structure and to evaluate their activity as antivirals.

Alkynes↗
PubMedDate not supplied

Activation of anti-reverse transcriptase nucleotide analogs by nucleoside diphosphate kinase: improvement by alpha-boranophosphate substitution.

Nucleoside activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively for a new class of nucleoside analogs with a borano (BH3-) or a thio (SH) group on the alpha-phosphate. Both the alpha-Rp-borano derivatives of AZT and d4T improved phosphorylation by NDP kinase, inhibition of reverse transcription as well as stability of alpha-borano nonophosphate derivatives in terminated viral DNA chain.

Anti-HIV Agents↗
PubMed1984

Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase.

The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.

Animals↗
PubMed2006

Design, synthesis and biological evaluations of novel oxindoles as HIV-1 non-nucleoside reverse transcriptase inhibitors. Part I.

A novel oxindole was discovered as an HIV non-nucleoside reverse transcriptase inhibitor via HTS using a cell-based assay. Systematic structural modifications were carried out to establish its SAR. These modifications led to the identification of oxindoles with low nanomolar potency for inhibiting HIV replication. These novel and potent oxindoles could serve as advanced leads for further optimizations.

Anti-HIV Agents↗
PubMed2005

Quantum computational analysis for drug resistance of HIV-1 reverse transcriptase to nevirapine through point mutations.

Quantum chemical calculation has been carried out to analyze binding interactions of nevirapine to HIV-1 reverse transcriptase (RT) and single point mutants Lys103 --> Asn (K103N) and Tyr181--> Cys (Y181C). In this study, the entire system of HIV-1 RT/nevirapine complex with over 15,000 atoms is explicitly treated by using a recently developed MFCC (molecular fractionation with conjugate caps) approach. Quantum calculation of protein-drug interaction energy is performed at Hartree-Fock and DFT levels. The RT-nevirapine interaction energies are computed at fixed geometries given by the crystal structures of the HIV-1 RT/nevirapine complexes from protein data bank (PDB). The present calculation provides a quantum mechanical interaction spectrum that explicitly shows interaction energies between nevirapine and individual amino-acid fragments of RT. Detailed interactions that are responsible for drug resistance of two major RT mutations are elucidated based on computational analysis in relation to the crystal structures of binding complexes. The present result provides a qualitative molecular understanding of HIV-1 RT drug resistance to nevirapine and gives useful guidance in designing improved inhibitors with better resistance to RT mutation.

Anti-HIV Agents↗
PubMed1993

Non-nucleoside inhibitors of HIV-1 reverse transcriptase: molecular modeling and X-ray structure investigations.

The structural features of a new class of non-nucleoside HIV-1 reverse transcriptase inhibitors (3) are presented. Comparison of the structural and electronic properties with those of TIBO (1) and Nevirapine (2) yields a common three-dimensional model. This model permits the improvement of the lead compound 3 by chemical modification (5,6). Additionally, two new types of inhibitors (4, 7) with similar biological activity can be derived from this model. The structure of the new compounds, including their absolute configuration, are determined by X-ray crystallography.

Benzodiazepines↗