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PubMed1989

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Animals↗

PubMed1978

Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus.

The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.

Avian Leukosis Virus↗

PubMed1994

Screening anti-HIV Chinese materia medica with HIV and equine infectious anemic virus reverse transcriptase.

The inhibitory activities of human immunodeficiency virus reverse transcriptase (HIV-RT) inhibitors PFA and Suramin against HIV-RT and equine infectious anemic virus reverse transcriptase (EIAV-RT) were studied in this paper. The 50% inhibitory concentration (IC50) of HIV-RT and EIAV-RT treated by PFA and Suramin were 0.2 mumol, 9.8 mumol and 17 mumol, 19.9 mumol, respectively. More than thirty Chinese medicines, including recipes, herbs, extracts of traditional materia medica and isolated compounds were tested using HIV-RT and EIAV-RT as target enzymes. It was found that 5 crude extracts such as Rhizoma Polygoni Cuspidati, and Radix Notoginseng, 7 isolated compounds like Flavone of Ramulus Visci, Flavone of Ajuga Decumbens Thumb, and Aristolochic acid, as well as the extract of the complex prescription Xiao Chai Hu Tang have shown various inhibitory actions on these two enzymes. The activities of the two enzymes were comparable, but HIV-RT was more sensitive, suggesting that HIV-RT can be used as index for the screening of anti-aids drugs.

Animals↗

PubMedDate not supplied

Expression pattern of telomerase reverse transcriptase in rectal carcinoma predicts tumor radiosensitivity, local recurrence and disease-free survival.

BACKGROUND/AIMS: Predictive indicators of radiosensitivity in rectal cancer are required in order to avoid unnecessary preoperative radiotherapy. Telomeres are specialized structures at the ends of chromosomes that protect against that such as irradiation. Lower telomerase activity has been reported to be related to radiosensitivity in vitro or vivo. The aim of this study was to examine the possible role of telomerase reverse transcriptase expression, which is correlated to telomerase activity, in determining tumor radiosensitivity, local recurrence, and disease-free survival in patients with rectal carcinomas. METHODOLOGY: We studied 96 cases with advanced rectal carcinoma. In preradiation biopsy specimens of tumor samples, the number of cells stained positive for telomerase reverse transcriptase protein was evaluated by immunohistochemistry. RESULTS: The expression pattern of telomerase reverse transcriptase protein correlated with tumor radiosensitivity (P=0.0007). All tumors that had a high percentage of telomerase reverse transcriptase positive cells were radioresistant. High telomerase reverse transcriptase expression pattern is a predictive marker for high local recurrence or poor survival in patients with rectal cancer (P=0.011, P=0.0005). Telomerase reverse transcriptase expression and histopathological grade were significant prognostic variables for disease-free survival in the multivariate analysis (P=0.0003, P=0.0091). CONCLUSIONS: Telomerase reverse transcriptase raises the predictive possibility of tumor radiosensitivity in rectal cancer. Telomerase reverse transcriptase might be a useful parameter for patient selection for preoperative radiotherapy in rectal carcinoma.

Adenocarcinoma↗

PubMed1986

Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli.

The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated an 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s).

AKR murine leukemia virus↗

PubMed1977

Purification, biochemical characterization and serological analysis of cellular deoxyribonucleic acid polymerases and a reverse transcriptase from spleen of a patient with myelofibrotic syndrome.

The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and template-primers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation-velocity measurements of the purified enzymes gave values of 150000, 40000, 100000 and 70000 daltons for DNA polymerase-alpha DNA polymerase-beta, DNA polymerase-gamma and the reverse transcriptase respectively. Serological studies have shown that the reverse transcriptase from human spleen is not antigenically related to cellular DNA polymerase-alpha, -beta or -gamma, but is antigenically related to reverse transcriptase from simian sarcoma virus and gibbon-ape leukaemia virus.

Child, Preschool↗

PubMed2002

A tight-binding mode of inhibition is essential for anti-human immunodeficiency virus type 1 virucidal activity of nonnucleoside reverse transcriptase inhibitors.

It was previously found that certain nonnucleoside reverse transcriptase inhibitors (NNRTI) possess virucidal activity against human immunodeficiency virus type 1 (HIV-1), and it was suggested that the tight-binding mode of inhibition of reverse transcriptase might be important for this virucidal activity (Borkow et al., J. Virol. 71:3023-3030, 1997). To test this, we compared six different NNRTI, including three tight-binding NNRTI, namely UC781, efavirenz (EFV) (Sustiva), and 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC), and three rapid-equilibrium NNRTI, delavirdine (DLV) (Rescriptor), nevirapine (NVP) (Viramune), and UC84, in a variety of virucidal tests. Incubation of isolated HIV-1 virions with UC781, EFV, or CSIC rapidly inactivated the virus, whereas DLV, NVP, and UC84 were ineffective in this respect. Exposure of H9+ cells chronically infected by HIV-1 to the tight-binding NNRTI abolished the infectivity of nascent virus subsequently produced by these cells following removal of extracellular drug, thereby preventing cell-to-cell virus transmission in the absence of exogenous drug. In contrast, cell-to-cell transmission of HIV was blocked by DLV, NVP, and UC84 only when the drug remained in the extracellular medium. Pretreatment of uninfected lymphocytoid cells with UC781, EFV, or CSIC, but not DLV, NVP, or UC84, protected these cells from subsequent HIV-1 infection in the absence of extracellular drug. The protective effect was dependent on both the dose of NNRTI and the viral load. The overall virucidal efficacy of the tight-binding NNRTI tested was CSIC > UC781 approximately EFV. We conclude that the tight-binding mode of inhibition is an essential characteristic for virucidal NNRTI and that antiviral potency is an insufficient predictor for virucidal utility of NNRTI.

Anti-HIV Agents↗

PubMed2001

Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue.

The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2',3'-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2',3'-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k(pol) mostly accounts for the lower incorporation efficiency observed for 2',3'-dideoxynucleotides. Examination of the crystal structure of the RT.DNA.dNTP complex suggested that both the charge at position 65 and the 3'-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2',3'-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3'-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. alpha-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.

Amino Acid Substitution↗

PubMed2005

Nonnucleoside reverse transcriptase inhibitor phenotypic hypersusceptibility can be demonstrated in different assays.

BACKGROUND: HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. OBJECTIVE: To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. METHODS: The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. RESULTS: Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. CONCLUSIONS: NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Amino Acid Substitution↗

PubMed1971

Distinguishing reverse transcriptase of an RNA tumor virus from other known DNA polymerases.

Assays are described that permit one to distinguish the reverse transcriptase of RNA tumor viruses from known normal cellular DNA-instructed DNA polymerases. Template responses of purified reverse transcriptase were compared with those of similar preparations of the DNA polymerase I of Escherichia coli and of calf-thymus polymerase. All three enzymes responded well to the synthetic duplexes poly(dT).poly(A), poly(U).poly(A), and poly(dT).poly(dA). Hence, these duplexes can detect, but cannot distinguish reverse, transcriptase from the known normal DNA polymerases. However, certain oligomer-homopolymer complexes serve as excellent distinguishing agents. The reverse transcriptase responds very well to (dT)(10).poly(A) and very poorly to (dT)(10).poly(dA), whereas both cellular DNA polymerases do not exhibit this behavior.Purified single-stranded RNA also serves as a diagnostic device, since only reverse transcriptase gives a detectable response. To be definitive, a positive response to RNA must be accompanied by a demonstration via molecular hybridization that the DNA product is complementary to the RNA and not to some minor DNA contaminant.

Adenine Nucleotides↗

PubMed1990

[Application of sensitive reverse transcriptase assay to human immunodeficiency virus (HIV) type 2].

Sensitive reverse transcriptase assay was applied to human immunodeficiency virus type 2. The kinetics of this assay, stability of the enzyme and the effect of BSA to this assay indicated that the condition of this assay should be 37 degrees C in reaction temperature. The sensitivity of this assay increased by adding more than 10 micrograms/ml of BSA. The sensitivity of this assay is at least four times more than that of CPE assay using Molt-4 cell. Other HIV-2 isolate, LAV-2 in culture medium was also detectable in this condition. Moreover reverse transcriptase inhibiting antibody that specifically inhibits HIV-2 reverse transcriptase was found by this assay.

HIV↗

PubMed2006

Developing novel nonnucleoside HIV-1 reverse transcriptase inhibitors: beyond the butterfly.

To date three nonnucleoside reverse transcriptase inhibitors (NNRTIs) have been approved by the U.S. Food and Drug Administration for the treatment of human immunodeficiency virus type 1 infection. A limiting factor in the effectiveness of these agents is the development of resistance, manifested by amino acid substitutions within the virally encoded reverse transcriptase (RT). Understanding the mechanism of action of these agents and how resistance develops have broadened the field of NNRTI research to elucidate structural and biochemical features of inhibition in hopes of creating better inhibitors. In this review, the history of NNRTIs will preface the many studies characterizing inhibition and the development of a new paradigm for understanding the molecular mechanism of drug resistance to NNRTIs. Combination therapies including nonnucleoside inhibitors will be discussed, concluding with remarks on potential new inhibitors.

Anti-HIV Agents↗

PubMed1999

Non-nucleoside reverse transcriptase inhibitors: the NNRTI boom.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are promising drugs for the treatment of HIV when used in combination with other anti-HIV drugs such as nucleoside reverse transcriptase (RT) inhibitors and protease inhibitors. The first generation of NNRTIs have, however, suffered from the rapid development of resistance. This review discusses the properties of the FDA-approved NNRTI drugs and focuses on the recent efforts being made to produce second generation inhibitors that circumvent this resistance problem.

Animals↗

PubMed1993

Monoclonal antibody-mediated inhibition of HIV-1 reverse transcriptase polymerase activity. Interaction with a possible deoxynucleoside triphosphate binding domain.

A series of monoclonal antibodies against p51/p66 human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT) were prepared by immunizing mice with the native enzyme immobilized on nitrocellulose. One of these antibodies, designated 1E8, was found to inhibit both RNA-dependent and DNA-dependent polymerase activities of RT but had no effect on the RNase H activity of the enzyme. This inhibition was noncompetitive with respect to primer/template and competitive with respect to deoxynucleoside triphosphate (dNTP). The extent of 1E8 inhibition of RT polymerase activity decreased with increasing concentrations of dNTP in the incubation but was not affected by changes in primer/template concentration. 1E8 bound equally well in solution to both free RT and to the RT-primer/template complex. However, binding to the latter was significantly reduced by the addition of increasing concentrations of dNTP. The ability of dNTP to inhibit the interaction of 1E8 with the RT-primer/template complex was dependent on the identity of the homopolymeric primer/template used; only that dNTP complementary to the template was effective in this respect. 1E8 bound to the p51/p66 reverse transcriptase heterodimer in solution and reacted with both p51 and p66 subunits of reverse transcriptase on Western blots. The antibody is therefore presumed to recognize a linear surface epitope on the enzyme. 1E8 was found to specifically recognize a peptide with the sequence KKDSTKWRK. This sequence corresponds to residues 65-73 of HIV-1 reverse transcriptase, a region identified as highly antigenic by several computer algorithms. Two mutations within this sequence have been identified with resistance to 3'-azido,3'-deoxythymidine. We conclude that residues 65-73 of HIV-1 reverse transcriptase may be at or near the polymerase active site of the enzyme, and may form part of the deoxynucleoside triphosphate binding domain of the enzyme.

Amino Acid Sequence↗

PubMed1993

Isolation and characterization of monoclonal antibodies raised against the reverse transcriptase of human immunodeficiency virus type 2 and cross-reactivity with that of type 1.

Monoclonal antibodies to human immunodeficiency virus (HIV)-2 reverse transcriptase have been raised with the ultimate goal of generating Fab fragments for future co-crystallization studies. A number of mouse monoclonal antibodies to recombinant HIV-2 reverse transcriptase have been obtained and characterized in terms of the possible epitopes they recognise together with cross-reactivity with a related reverse transcriptase. The antibodies were shown to fall into three groups that recognize different regions of the reverse transcriptase enzyme. One antibody, which recognizes part of the RNase H domain, demonstrated cross-reactivity between the HIV-1 and HIV-2 reverse transcriptase.

Amino Acid Sequence↗

PubMed1989

Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase.

We have described a strain of Escherichia coli that expresses high levels of enzymatically active, soluble, HIV-1 reverse transcriptase (A. Hizi, C. McGill, and S. H. Hughes, Proc. Natl. Acad. Sci. USA, 85, 1218-1222, 1988). The clone can be used as a source of the enzyme and to generate and characterize mutations in the reverse transcriptase. We have made a series of small in-frame insertions in the region that encodes the reverse transcriptase. When the mutant plasmids are reintroduced into E. coli, they induce the synthesis of mutant forms of the enzyme. With one interesting exception, the reduction in RNA-dependent DNA polymerizing activity seen in the mutants correlates well with the degree of sequence conservation among the various reverse transcriptases. Insertions into regions that are evolutionarily conserved have a more profound effect on RNA-dependent DNA polymerase activity than do insertions into regions that are less conserved. The exception to this simple correlation is that a small insertion into the region encoding RNase H gives rise to a protein with essentially no RNA-dependent DNA polymerase activity. We suggest that this mutation may affect the ability of the reverse transcriptase to fold properly, which might explain our previous observation that small carboxyl terminal deletions profoundly affect RNA-dependent NAD polymerase activity.

Binding Sites↗

PubMed1994

Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution.

Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.

Crystallography, X-Ray↗

PubMed2005

Mutagenic properties of ribonucleotide analogues in reverse transcription with HIV and AMV reverse transcriptases.

Pyrimidine analogues, N4-hydroxycytosine (C(oh)), N4-methoxycytosine (C(mo)) and 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P) can form base pairs with both adenine and guanine. We examined the mutagenic properties of these ribonucleotide analogues in RNA in reverse transcription with HIV and AMV reverse transcriptases. Both reverse transcriptases incorporated dATP and dGTP opposite these analogues in RNA. The incorporation ratio of dGTP to dATP opposite each analogue was measured to estimate the potential for inducing U-to-C mutations. The potentials may be rC(oh) > rC(mo) > rP for both reverse transcriptases. It might be possible to induce mutations in the retroviral genomes and to develop a new antiviral therapy, if these analogues are incorporated by a human RNA polymerase.

Avian Myeloblastosis Virus↗