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PubMed2004

Nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

The development of novel compounds that can effectively inhibit both wild type and the most consensus resistant strains of human immunodeficiency virus type 1 (HIV-1) is the primary focus in HIV disease management. Combination therapy, comprising at least three classes of drugs, has become the standard of care for acquired immunodeficiency syndrome (AIDS) or HIV-infected individuals. The drug cocktail can comprise all three classes of HIV inhibitors, including nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Due to their competitive mode of inhibition and requirement for metabolic activation, almost all NRTI drugs lack the virological potency of NNRTI or PI drugs. However, data from clinical trials indicate that sustained viral suppression could not be achieved with NRTI, NNRTI or PIs alone. Therefore, the NRTIs will remain essential components of highly active antiretroviral therapy (HAART) for the foreseeable future, because they enhance the virological potency of the regimen, they do not bind excessively to protein and most regimens are small pills/tablets given once a day. It has become apparent in recent years that the prolonged use of certain NRTIs exhibits adverse events as a class, limiting the length of time for which they can be safely used. Of major clinical concern is their association with the potentially fatal lactic acidaemia and hepatic steatosis. These class events, as well as individual drug effects, such as peripheral neuropathy, are linked to delayed mitochondrial destruction. In addition to toxicity, the development of resistance-conferring mutations against exposure to nucleoside analogs currently in use influences long-term therapeutic benefits. Of critical importance for the evaluation of new NRTIs are recent studies showing that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway.

Anti-HIV Agents↗

PubMed1998

p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.

Base Sequence↗

PubMed2001

Human Immunodeficiency Virus Reverse Transcriptase and Protease Sequence Database: an expanded data model integrating natural language text and sequence analysis programs.

The HIV Reverse Transcriptase and Protease Sequence Database is an on-line relational database that catalogs evolutionary and drug-related sequence variation in the human immunodeficiency virus (HIV) reverse transcriptase (RT) and protease enzymes, the molecular targets of anti-HIV therapy (http://hivdb.stanford.edu). The database contains a compilation of nearly all published HIV RT and protease sequences, including submissions from International Collaboration databases and sequences published in journal articles. Sequences are linked to data about the source of the sequence sample and the antiretroviral drug treatment history of the individual from whom the isolate was obtained. During the past year 3500 sequences have been added and the data model has been expanded to include drug susceptibility data on sequenced isolates. Database content has also been integrated with didactic text and the output of two sequence analysis programs.

Amino Acid Sequence↗

PubMed1988

Conformation and sandwiching of bases by azido groups in the crystal structure of 3'-azido-3'-deoxy-thymidine (AZT), an antiviral agent that inhibits HIV reverse transcriptase.

The crystal structure of 3'-azido-3'-deoxy-thymidine (AZT), an antiviral agent that inhibits HIV reverse transcriptase, has been determined from three-dimensional x-ray diffractometer data. The crystal structure contains two independent molecules of AZT forming a hydrogen bonded dimer but exhibiting different conformations. These conformations are different from those theoretically calculated by molecular mechanics methods. The azido groups associate with each other and interrupt the base stacking, forming a sandwich of two stacked bases. The close conformational similarity of AZT to thymidine explains why AZT is a good substrate for thymidine kinase. The selective inhibition of reverse transcriptase by AZT is not due to any conformational restrictions imposed by the azido group but likely due to their stereoelectronic properties.

Azides↗

PubMed2001

Phenotypic and genotypic resistance to nucleoside reverse transcriptase inhibitors in HIV-1 clinical isolates.

OBJECTIVE: To assess phenotypic and genotypic cross-resistance to nucleoside reverse transcriptase inhibitors in patients treated with a combination including zidovudine, who were switched to a combination including stavudine. METHODS: We analysed 24 clinical HIV-1 isolates from 12 patients before and several months after therapeutic switching. Plasma HIV-1 RNA was measured using quantitative polymerase chain reaction (Roche). Genotypic resistance was measured by sequencing the reverse transcriptase gene from plasma HIV-1 RNA. Phenotypic resistance was measured using a recombinant assay (Virco). RESULTS: Patients were treated with a combination including zidovudine for a mean (+/- SEM) period of 21.8 +/- 3.5 months and had a plasma viral load of 4.1 +/- 0.2 log HIV-1 RNA copies/mL (time 1). After a mean period of 19.3 +/- 1.6 months following the therapeutic change, the plasma viral load was 3.6 +/- 0.1 log copies/mL (time 2). At time 1, genotypic resistance to zidovudine was found in all cases (41L: four cases; 41L, 215Y: five cases; 41L, 210W, 215Y: two cases; K70R: one case) with a mean 6.6 +/- 1.6-fold increase in the median inhibitory concentration (IC50) to zidovudine and 1.7 +/- 0.4-fold to stavudine. At time 2, genotypic resistance to zidovudine was found in 11 out of 12 cases (41L: two cases; 41L, 215Y: six cases; 41L, 210W, 215Y: two cases; M41L, D67N, L210W, T215Y: one case) with a mean 18.9 +/- 8.8-fold increase in the IC50 to zidovudine and 1.4 +/- 0.4-fold to stavudine CONCLUSIONS: In this clinical series of patients with suboptimal control of plasma HIV-1 RNA using a combination including zidovudine, the presence of zidovudine-related mutations was associated with a decreased phenotypic sensitivity to this drug. Despite persistent HIV-1 replication, switching to stavudine was associated with a further decrease in phenotypic sensitivity to zidovudine but not to stavudine after 19 months. These data suggest that stavudine remains a possible option in zidovudine-experienced patients.

Adult↗

PubMed2006

High rate multiple drug resistances in HIV-infected patients failing nonnucleoside reverse transcriptase inhibitor regimens in Thailand, where subtype A/E is predominant.

The prevalence of drug resistance was determined among 64 HIV-infected Thai patients who were failed while receiving nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens. Eighty-nine percent of patients had 1 or more NNRTI mutation resistances. Almost all patients had resistance to at least 1 nucleoside reverse transcriptase inhibitor (NRTI), and 42% had multiple-NRTI resistance.

Adult↗

PubMed1992

Expression of chimeric proteins encoded by the fused BLV reverse transcriptase: beta-galactosidase in Escherichia coli.

The gene for bovine leukemia virus (BLV) reverse transcriptase was cloned in prokaryotic expression vector pUC8-2. After fusion of Escherichia coli lacZ gene to different parts of reverse transcriptase we detected expression of new proteins with molecular weights corresponding to the size of the hybrid genes. A coding region most probably responsible for about a hundred-fold decrease in expression of long fusion proteins has been identified. A few possible causes of this phenomenon were tested.

Escherichia coli↗

PubMed2004

Closing of the fingers domain generates motor forces in the HIV reverse transcriptase.

Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (approximately 15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.

Binding Sites↗

PubMed1986

Inhibition by sakyomicin A of avian myeloblastosis virus reverse transcriptase and proliferation of AIDS-associated virus (HTLV-III/LAV).

In the course of screening for inhibitors of reverse transcriptase, we have isolated an inhibitor from a strain of Nocardia and identified it as sakyomicin A. The antibiotic blocks avian myeloblastosis virus reverse transcriptase reaction: IC50 was ca. 30 micrograms/ml by the method employed. The drug affects proliferation of HTLV-III/LAV in HTLV-I-carrying MT-4 cells: ca. 60% inhibition was observed at an antibiotic concentration of 1.0 microgram/ml and ca. 20% inhibition at 0.1 microgram/ml, and there was no significant cytotoxicity.

Avian Leukosis Virus↗

PubMed2003

Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates.

Nucleoside reverse-transcriptase inhibitors (NRTIs) are important components of most antiretroviral combination treatment regimens. Using a large collection of clinical isolates, we characterized patterns of cross-resistance among all NRTIs. Drugs were grouped by the effect of the M184V mutation: susceptibility to group 1 drugs (zidovudine, stavudine, tenofovir, and adefovir) increased when M184V was present, whereas susceptibility to group 2 drugs (didanosine, zalcitabine, abacavir, and lamivudine) decreased. Significant cross-resistance was observed among all NRTIs and was most notable when samples with or without M184V were analyzed separately. An increasing number of thymidine-analogue mutations (TAMs) was associated with a progressive reduction in drug susceptibility for all NRTIs. The modulating effect of M184I/V on drug susceptibility was present regardless of the number of TAMs. The broad range of susceptibility observed for viruses containing the same number of TAMs indicates that the genetic correlates of NRTI resistance remain to be fully elucidated.

Anti-HIV Agents↗

PubMed1996

RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity.

The reverse transcriptase-associated RNase H activity of Moloney murine leukemia virus specifically cleaves within the polypurine tract region of the viral genome to generate the primer for plus-strand DNA synthesis and removes the tRNA primer after minus-strand initiation by preferentially cleaving the RNA one nucleotide before the RNA-DNA junction. Moreover, the enzyme is unable to cleave the extended tRNA substrate at the RNA-DNA junction even at high enzyme concentrations. The RNase H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extracts. Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determined in the plus-strand primer reaction and in the tRNA primer removal reaction. Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA-DNA junction. Specificity in both cases could be restored by adding back a truncated form of Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain. These results implicate the polymerase domain as a specificity determinant for the RNase H activity of reverse transcriptase. The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RNase H domain alone nor the RNase H domain coupled to the polymerase domain in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation.

DNA-Directed DNA Polymerase↗

PubMed1991

Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase.

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain.

Amino Acid Sequence↗

PubMed1996

Human endogenous retrovirus expression and reverse transcriptase activity in the T47D mammary carcinoma cell line.

We have examined the human mammary tumor cell line T47D and have found that these cells produce virus-like particles which band at the typical density for retroviral particles on a sucrose gradient, possess reverse transcriptase activity, and package HERV-K10-like sequences. Using this information and a bacterial expression system to identify long open reading frames, we have identified individual clones which have full-length open reading frames for reverse transcriptase and RNase H and which could encode the reverse transcriptase activity detected in these cells.

Base Sequence↗

PubMed2003

Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template.

In order to identify the reverse transcriptase activity in sera and conditioned media from peripheral blood mononuclear cells (PBMCs) of large granular lymphocyte leukemia patients product enhanced reverse transcriptase activity (PERT) assays were performed using bacteriophage MS2 RNA as a template. All samples obtained from conditioned media of virus-infected cell lines as well as PBMCs of lymphocytic leukemia patients and normal healthy individuals tested positive with this assay. Therefore the validity of the assay was questioned. Careful evaluation of the assay revealed that some of the essential reagents used, such as Taq DNA polymerase and RNase inhibitor contained indigenous amplifiable DNA. DNase I treatment of Taq DNA polymerase before PCR reduced the product significantly. Moreover, no false positive results were observed when encephalomyocarditis virus RNA was used instead of MS2 RNA as the template. These results suggest a need for caution when using bacteriophage MS2 RNA as the template in PERT assays to confirm the presence of retroviral infection or for identification of novel retroviruses.

Humans↗

PubMed1983

Inhibition of reverse transcriptases by seminalplasmin.

Seminalplasmin, an antibacterial protein present in bovine seminal plasma, is shown to be a potent inhibitor of reverse transcriptases (RNA-dependent DNA nucleotidyltransferases). Seminalplasmin inhibits RNA-directed, hybrid-directed, and DNA-directed DNA-polymerizing activities of purified reverse transcriptase from avian myeloblastosis virus and from crude viral lysates of several retroviruses by binding to the enzyme, at least in the case of avian myeloblastosis virus. Seminalplasmin does not inhibit significantly DNA synthesis either by Escherichia coli DNA polymerase I, or a mammalian alpha-DNA polymerase. The presence of seminalplasmin in the seminal fluid could provide protection to the male and/or the female reproductive tract against retroviruses.

Avian Myeloblastosis Virus↗

PubMed1998

An enzymatically active chimeric HIV-1 reverse transcriptase (RT) with the RNase-H domain of murine leukemia virus RT exists as a monomer.

The existence of retroviral reverse transcriptases as monomers or dimers is rather intriguing. A classical example of the former is murine leukemia virus reverse transcriptase (MuLV RT), while human immunodeficiency virus type 1 (HIV-1) RT represents the latter. A careful scrutiny of the amino acid sequence alignment of the two enzymes pinpoints the region tentatively responsible for this phenomenon. We report here the construction of a chimeric enzyme containing the first 425 amino acid residues from the N-terminal domain of HIV-1 RT and 200 amino acid residues from the C-terminal domain of MuLV RT. The chimeric enzyme exists as a monomer with intact DNA polymerase and RNase-H functions.

Amino Acid Sequence↗

PubMed2006

Correlation between radiation-induced telomerase activity and human telomerase reverse transcriptase mRNA expression in HeLa cells.

PURPOSE: To quantify and correlate human telomerase reverse transcriptase (hTERT) mRNA expression with telomerase activity (TA) after ionizing irradiation of HeLa cells. MATERIALS AND METHODS: TA and hTERT mRNA expression were evaluated, at 24-h intervals, in HeLa cells cultured for up to 144 h, before and after treatment with increasing doses of 6 MV photon ionizing radiation (5 - 20 Gy), using the telomeric repeat amplification protocol (TRAP) assay and real-time reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Cell viability was determined using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. A prototype phantom was constructed for accurate irradiation of HeLa cells. RESULTS: Treated cells showed a decrease in viability with increasing radiation dose, and a correlation was observed with post-treatment period. TA and hTERT mRNA expression of HeLa cells increased for the first 24 h after irradiation. The maximal increases were approximately two times the un-irradiated cell levels at 24 h post-irradiation, followed by a decrease and a return to the control levels 72 h post-irradiation. The time-course of telomerase activation after 24 h, differed among radiation doses. A dose-dependent G2/M arrest was observed 24 h post-irradiation, along with an increase in polyploidy 48 h post-irradiation and afterwards. CONCLUSION: A correlation between TA and hTERT mRNA expression and a radiation induced cell cycle dependent modification of hTERT mRNA expression was established for the first 24 h post-irradiation.

Cell Survival↗

PubMed2002

A highly sensitive chemiluminescent reverse transcriptase assay for human immunodeficiency virus.

A simple and highly sensitive reverse transcriptase (RT) assay was developed by combining a previously reported non-radioisotopic RT assay with the use of a template-primer-immobilized microplate, an enzyme capture protocol, product digestion and a chemiluminescent substrate. The assay was able to detect directly the RT activity in serum samples, plasma and cell culture medium without the need for concentration and extraction of the enzyme. The assay was able to detect RT activity equivalent to 100 virions/ml of HIV-1. These results suggest that this highly sensitive chemiluminescent RT assay can be used not only for virological investigation but also for routine screening of biopharmaceuticals.

Cells, Cultured↗