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PubMed1995

Nevirapine alters the cleavage specificity of ribonuclease H of human immunodeficiency virus 1 reverse transcriptase.

The action of the dipyridodiazepinone nevirapine (BI-RG-587) on polymerization and RNase H activities of human immunodeficiency virus reverse transcriptase (RT) was examined. Substrates using heteropolymeric DNA primers hybridized to complementary RNA templates were employed. Challenged assays were performed that allowed measurement of activity of the RT resulting from a single round of binding of RT to substrate. Results demonstrated that nevirapine alters the cleavage specificity of the RNase H. Instead of a primary cleavage approximately 18 nucleotides upstream of the DNA 3' terminus, multiple cleavages were observed ahead of and behind this site. This indicated that the compound facilitates sliding of the RT away from the DNA primer terminus allowing cleavage at more sites. The change in specificity occurred whether the primer terminus was at the end or internal on the template. Experiments with RNA primers on circular DNA demonstrated a nevirapine-induced stimulation of RNase H activity beyond the increase expected from the change in cleavage specificity. Examination of polymerization showed that the compound decreased both the number of primers that underwent synthesis and the processive elongation of those primers. The significance of these results with respect to viral replication and recombination is discussed.

Base Sequence↗
PubMed1992

Quinolinehydrazones as inhibitors of retroviral reverse transcriptase.

The inhibitory activity of a series of 2- and 4-quinolinehydrazones on retroviral reverse transcriptase has been studied on enzymes from M-MuLV, RAV-2, and on a crude lysate of HIV-1, assuming the first two enzymes as potential models of the third. The highest activity is mainly found in lipophilic, water soluble 4-quinolinehydrazones. The inhibitory activity of these compounds decreases in changing from the M-MuLV to the RAV-2, and HIV-1 enzymes, in this order.

Cell Survival↗
PubMed1996

Comparative kinetic analyses of interaction of inhibitors with Rauscher murine leukemia virus and human immunodeficiency virus reverse transcriptases.

The inhibitory effects of several nucleoside triphosphate analogs on Rauscher murine leukemia virus (RMuLV) and human immunodeficiency virus (HIV) type 1 reverse transcriptases (RTs) were studied. With RNA as the template, the apparent K(m) and apparent K(i) values of HIV RT toward its substrates and inhibitors are 12 to 500 times lower than the corresponding values for RMuLV RT. However, the k(i)/k(m) ratios (inhibition efficiencies) for HIV and RMuLV RTs'are similar for AZTTP (zidovudine triphosphate), d4TTP [3'-deoxythymidine-2'-ene-(3'-deoxy-2',3'-didehydrothymidine) triphosphate], PMEADP [9-(2-phosphonylmethoxyethyl)adenine diphosphate], FIAUTP [1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodouracil triphosphate], and HPMPCDP [(S)-1-(3-hydroxy-2-phosphylmethoxypropyl) cytosine diphosphate]. With DNA as the template, the K(m) values are similar for HIV and RMuLV RTs. However, the K(i)/K(m) values of HIV and RMuLV RTs are significantly different for ddCTP, ddATP, and 3TCTP (2',3'-dideoxy-3'-thiacytidine). The RTs of RMuLV and HIV are sufficiently different from one another that the kinetic inhibition constants for a particular antiviral compounds should be determined to indicate whether anti-RMuLV activity is likely to be predictive for the anti-HIV activity of the compound. This information, in conjunction with species-specific drug metabolism differences and tissue culture antiviral activity, is important in determining the suitability of a particular animal model.

Adenine↗
PubMed1997

Highly potent oxathiin carboxanilide derivatives with efficacy against nonnucleoside reverse transcriptase inhibitor-resistant human immunodeficiency virus isolates.

The structure-activity relationships of a series of compounds related to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) oxathiin carboxanilide have been described (R. W. Buckheit, Jr., T. L. Kinjerski, V. Fliakas-Boltz, J. D. Russell, T. L. Stup, L. A. Pallansch, W. G. Brouwer, D. C. Dao, W. A. Harrison, R. J. Schultz, J. P. Bader, and S. S. Yang, Antimicrob. Agents Chemother. 39:2718-2727, 1996). From these studies, the furanyl-containing analog UC10 was identified as the most potent inhibitor of human immunodeficiency virus type 1 (HIV-1) replication and a promising candidate for further development. Three new UC analogs (UC040, UC82, and UC781) have been determined to inhibit laboratory-derived and low-passage-number, primary virus isolates at low nanomolar concentrations in both established and fresh human cells. Each of the compounds synergistically interacted with the nucleoside analogs zidovudine, dideoxyinosine, dideoxycytosine, and lamivudine to inhibit HIV-1 replication. As a group, the UC compounds were found to be less active against viruses with the L100I, K103N, and Y181C amino acid changes in the RT and, upon in vitro selection, yielded resistant virus with the Y181C mutation in the RT. The most potent of the three new compounds, UC781, contains a furanyl side chain, similar to UC10, but differs in having an extended ether side chain instead of an oxime chain. The broad therapeutic index of UC781 (>62,000) resulted in effective inhibition of NNRTI-resistant virus isolates at high nanomolar concentrations. Furthermore, UC781 and the NNRTI costatolide were able to synergistically inhibit HIV-1 replication when used in combination, suggesting that UC781 may interact with the RT differently than the other UC analogs. The favorable anti-HIV properties of the UC compounds suggest they should be considered for further clinical development.

Anti-HIV Agents↗
PubMed2006

Synthesis and biological evaluations of sulfanyltriazoles as novel HIV-1 non-nucleoside reverse transcriptase inhibitors.

A novel sulfanyltriazole was discovered as an HIV-1 non-nucleoside reverse transcriptase inhibitor via HTS using a cell-based assay. Chemical modifications and molecular modeling studies were carried out to establish its SAR and understand its interactions with the enzyme. These modifications led to the identification of sulfanyltriazoles with low nanomolar potency for inhibiting HIV-1 replication and promising activities against selected NNRTI resistant mutants. These novel and potent sulfanyltriazoles could serve as advanced leads for further optimization.

Anti-HIV Agents↗
PubMed2002

Prediction of activity for nonnucleoside inhibitors with HIV-1 reverse transcriptase based on Monte Carlo simulations.

Results of Monte Carlo (MC) simulations for more than 200 nonnucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) representing eight diverse chemotypes have been correlated with their anti-HIV activities in an effort to establish simulation protocols and methods that can be used in the development of more effective drugs. Each inhibitor was modeled in a complex with the protein and by itself in water, and potentially useful descriptors of binding affinity were collected during the MC simulations. A viable regression equation was obtained for each data set using an extended linear response approach, which yielded r(2) values between 0.54 and 0.85 and an average unsigned error of only 0.50 kcal/mol. The most common descriptors confirm that a good geometrical match between the inhibitor and the protein is important and that the net loss of hydrogen bonds with the inhibitor upon binding is unfavorable. Other physically reasonable descriptors of binding are needed on a chemotype case-by-case basis. By including descriptors in common from the individual fits, combination regressions that include multiple data sets were also developed. This procedure led to a refined "master" regression for 210 NNRTIs with an r(2) of 0.60 and a cross-validated q(2) of 0.55. The computed activities show an rms error of 0.86 kcal/mol in comparison with experiment and an average unsigned error of 0.69 kcal/mol. Encouraging results were obtained for the predictions of 27 NNRTIs, representing a new chemotype not included in the development of the regression model. Predictions for this test set using the master regression yielded a q(2) value of 0.51 and an average unsigned error of 0.67 kcal/mol. Finally, additional regression analysis reveals that use of ligand-only descriptors leads to models with much diminished predictive ability.

Alkynes↗
PubMed2002

The potential role of rattus rattus in enzootic cycle of Rift Valley Fever in Egypt 2-application of reverse transcriptase polymerase chain reaction (RT-PCR) in blood samples of Rattus rattus.

A reverse transcriptase -polymerase chain reaction (RT-PCR) was applied to detect Rift Valley Fever Virus (RVF-V) in blood samples of Rattus rattus (R. rattus) collected from 3 different governorates of Egypt, Alexandria, Behira and Minia governorates (one hundred each). Out of 300 blood samples 29(9.67%) were positive for RVF-Virus by RT-PCR with higher percent in Behira governorate rural areas (16%), followed by Minia governorate rural areas (13.85%) while the lowest percent was in Alexandria governorate urban areas (0.00%). The overall percent in rural areas were (13.5%) while it was only (2.0%) in urban areas. Our Study suggests that, this R. rattus play an important role in the maintenance cycle of RVF-V in rural areas of Egypt.

Animals↗
PubMed2001

High degree of interlaboratory reproducibility of human immunodeficiency virus type 1 protease and reverse transcriptase sequencing of plasma samples from heavily treated patients.

We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself.

Amino Acid Sequence↗
PubMed2005

Reverse transcriptase inhibitors down-regulate cell proliferation in vitro and in vivo and restore thyrotropin signaling and iodine uptake in human thyroid anaplastic carcinoma.

CONTEXT: Two classes of repeated genomic elements, retrotransposons and endogenous retroviruses, encode for endogenous nontelomeric reverse transcriptase (RT), a gene that is down-regulated in differentiated cells but is highly expressed in embryonic and transformed tissues. Two nonnucleosidic RT inhibitors, efavirenz and nevirapine, currently used in HIV treatment, reversibly down-regulate tumor growth and induce differentiation in several human tumor cell models. OBJECTIVES: Aggressive biological behavior and loss of specific thyroid cell functions, such as thyroglobulin, thyroid peroxidase, TSH receptor, Na/I symporter expression, and iodine uptake are features of anaplastic thyroid cancer. Thus, we evaluated the use of RT inhibitors as a potentially differentiating and molecular-targeted treatment of this neoplasm. RESULTS: Our findings showed that nevirapine and efavirenz reversibly inhibit cell proliferation without triggering cell death in the undifferentiated thyroid carcinoma ARO and FRO cells, which exhibited high levels of endogenous RT activity. Inhibition of cell growth was correlated with accumulation of cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the S phase. Moreover, treated cells demonstrated a differentiated phenotype and a significant reprogramming of gene expression characterized by up-regulation of the TSH receptor, thyroglobulin, thyroid peroxidase, and Na/I symporter genes. Interestingly, RT inhibition reestablished the ability to uptake iodine in response to TSH either in vitro or in vivo and reversibly down-regulated tumor growth in mice xenografts of ARO cells. CONCLUSIONS: These findings support the need for clinical trials to clarify whether RT inhibitors may restore the sensitivity to radiometabolic therapy in anaplastic thyroid tumors.

Animals↗
PubMed1994

An active recombinant p15 RNase H domain is functionally distinct from the RNase H domain associated with human immunodeficiency virus type 1 reverse transcriptase.

An active p15 RNase H domain, consisting of amino acids 427-560 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and a genetically engineered penta-histidine N-terminal affinity tag, was expressed in Escherichia coli and purified to apparent homogeneity by immobilized metal affinity chromatography. The purified p15 RNase H domain exhibited no substrate preference for [3H]poly(rG).poly(dC) compared to [3H]poly(rA).poly(dT), in contrast with the HIV-1 RT-associated RNase H, which showed a 30-fold preference for the former substrate. Unlike the HIV-1 RT-associated RNase H, when challenged with unlabeled substrate, the recombinant p15 RNase H domain was relatively nonprocessive in RNA degradative activity of the [3H]poly(rA).poly(dT) duplex. Kinetic studies using p15 RNase H showed substrate inhibition with an apparent K(i) value of 0.12 micron for the [3H]poly(rA).poly(dT) hybrid. Substrate inhibition was not observed for the HIV-1 RT-associated RNase H. The results show that the isolated p15 HIV-1 RNase H domain is functionally distinct from the recombinant HIV-1 RT-associated RNase H.

Amino Acid Sequence↗
PubMed2004

Epigenetic regulation of human telomerase reverse transcriptase promoter activity during cellular differentiation.

The human telomerase reverse transcriptase (TERT) gene is transcriptionally inactivated in most differentiated cells but is reactivated in the majority of cancer cells. To elucidate how TERT is inactivated during differentiation, we applied all-trans retinoic acid (ATRA) to induce the differentiation of human teratocarcinoma (HT) cells and human acute myeloid leukemia (HL60) cells. We first showed that TERT promoter activity decreased rapidly, which preceded a gradual loss of endogenous telomerase activity following ATRA induction. To elucidate the underlying mechanisms of the reduced TERT promoter activity during differentiation, we performed epigenetic studies on the TERT promoter and found a progressive histone hypoacetylation coupled with a gradual accumulation of methylated cytosines in the TERT promoter. We also observed that the TERT promoter was less methylated in pluripotent HT cells than in multipotent HL60 cells throughout a 12-day differentiation process. This origin-dependent epigenetic change was also confirmed in histone acetylation studies, indicating that the TERT promoter was more resistant to deacetylation in HT cells than in HL60 cells. Taken together, our results demonstrate synergistic involvement of DNA methylation and histone deacetylation in the down-regulation of TERT promoter activity that may be dependent on the origin of the cell types, and they add new insight into the way telomerase activity may be regulated during differentiation.

Acetylation↗
PubMed1997

Genetic selection in Escherichia coli for active human immunodeficiency virus reverse transcriptase mutants.

Most catalytically active human immunodeficiency virus (HIV) reverse transcriptase (RT) mutants characterized to date have been isolated from the virus after treatment with HIV RT inhibitors such as nucleoside analogs. However, detailed understanding of structure-function relationships, and of the roles of the several catalytic activities of HIV RT in viral replication, requires characterization of a greater diversity of mutant enzymes than has been obtained from viral variants. Coupling of a bacterial genetic selection system for functional HIV RT with random mutagenesis has yielded a large number of active mutant enzymes, most of which have not been found in viral variants. The genetic selection system, combined with biochemical characterization of active mutant proteins, affords three major benefits. First, we can increase our understanding of the roles of individual amino acids in catalysis. Second, the mutational spectrum observed among active HIV RT variants can identify amino acids that are intolerant, or relatively intolerant, of substitution. Third, this system provides us with HIV RT variants with altered biochemical properties, such as replicational fidelity and processivity. Characterization of HIV harboring these mutant RTs with defined structural and functional alterations will contribute to elucidation of the roles of each catalytic activity of HIV RT in viral replication.

Chromatography, Affinity↗
PubMed1992

Contribution of the p51 subunit of HIV-1 reverse transcriptase to enzyme processivity.

Human immunodeficiency virus Type I reverse transcriptase is active as either the homodimer (p66/p66) or the heterodimer (p66/p51). Purified recombinant p66 and p51 expressed in yeast were reconstituted in the presence of 60 mM sodium pyrophosphate to enhance dimer formation. Comparison of the processivity of these two active reconstituted forms shows that the heterodimer is more processive than the homodimer with a cycle almost twice as long as judged by assays utilizing poly (U,G) as a challenger to primer-template. Binding assays demonstrated that the heterodimer has a higher affinity for primer-template than the homodimer and that the p51 subunit has an affinity equal to that of the heterodimer. These results suggest that the p51 subunit functions to increase processivity in the heterodimer.

Cloning, Molecular↗
PubMed2000

Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase.

We report the first use of human telomerase reverse transcriptase (hTERT) expression in experimental xenotransplantation. Previously, we showed that bovine adrenocortical cells can be transplanted into severe combined immunodeficient (SCID) mice, and that these cells form functional tissue that replaces the animals' own adrenal glands. We cotransfected primary bovine adrenocortical cells with plasmids encoding hTERT, SV40 T antigen, neo, and green fluorescent protein. These clones do not undergo loss of telomeric DNA and appear to be immortalized. Two clones were transplanted beneath the kidney capsule of SCID mice. Animals that received cell transplants survived indefinitely despite adrenalectomy. The mouse glucocorticoid, corticosterone, was replaced by the bovine glucocorticoid, cortisol, in the plasma of these animals. The tissue formed from the transplanted cells resembled that formed by transplantation of cells that were not genetically modified and was similar to normal bovine adrenal cortex. The proliferation rate in tissues formed from these clones was low and there were no indications of malignant transformation.

Adrenal Cortex↗
PubMed2004

Human DNA polymerase-eta, an A-T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase.

We have proposed previously that error-prone reverse transcription using pre-mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase-eta exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y-family polymerase is a reverse transcriptase (RT). This possibility was tested using a product-enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27-37 nucleotides. Human pol-eta and two other Y-family enzymes that are dispensable for SHM, human pols-iota and -kappa, copied a heteropolymeric DNA-primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol-eta was confirmed using a second sample from an independent source. Human DNA pols-beta and -mu, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol-eta was the most efficient RT of the Y-family enzymes assayed but was much less efficient than an HIV-RT standard in vitro. It is thus feasible that pol-eta acts as both a RNA- and a DNA-dependent DNA polymerase in SHM in vivo, and that Y-family RT activity participates in other mechanisms of physiological importance.

Base Sequence↗
PubMed2002

Human papillomavirus E2 down-regulates the human telomerase reverse transcriptase promoter.

The transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene is a critical step in transformation and differentiation. Human papillomavirus E2 protein inhibits cell growth in HPV-infected cells and triggers apoptosis in HeLa cells. Because E2 induces cell growth suppression and senescence, we hypothesize that the protein may modulate cellular gene expression related to these processes. In this report, we demonstrate that E2 inhibits the hTERT promoter. The mapping of the E2-responsive region of hTERT reveals that Sp1 is important for E2-mediated repression of this promoter in 293T cells. Site-directed mutagenesis data on the hTERT promoter show that E2 does not abolish E-Box-mediated transcription and represses promoter activity via the Sp1 binding site. Furthermore, chromatin immunoprecipitation assays indicate that E2 is actively recruited to the hTERT promoter region. Our findings provide novel insights into the biological function of human papillomavirus E2.

Animals↗
PubMed2004

Insights into the role of an active site aspartate in Ty1 reverse transcriptase polymerization.

Long terminal repeat-containing retrotransposons encode reverse transcriptases (RTs) that replicate their RNA into integratable, double-stranded DNA. A mutant version of the RT from Saccharomyces cerevisiae retrotransposon Ty1, in which one of the three active site aspartates has been changed to asparagine (D211N), is still capable of in vitro polymerization, although it is blocked for in vivo transposition. We generated recombinant WT and D211N Ty1 RTs to study RT function and determine specific roles for the Asp(211) residue. Presteady-state kinetic analysis of the two enzymes shows that the D211N mutation has minimal effect on nucleotide binding but reduces the k(pol) by approximately 230-fold. The mutation reduces binding affinity for both Mn(2+) and Mg(2+), indicating that the Asp(211) side chain helps create a tight metal binding pocket. Although both enzymes are highly processive and tend to remain bound to their initial substrate, each shows distinctive patterns of pausing, attributable to interactions between metal ions and the active site residue. These results provide insights to specific roles for the Asp(211) residue during polymerization and indicate unusual enzymatic properties that bear on the Ty1 replication pathway.

Aspartic Acid↗
PubMed2005

Two purified domains of telomerase reverse transcriptase reconstitute sequence-specific interactions with RNA.

Telomerase reverse transcriptase (TERT) and telomerase RNA (TER) function together to create a uniquely specialized polymerase. Here we have described for the first time domains of bacterially expressed Tetrahymena TERT that interacted directly with TER in the absence of assembly chaperones. We used quantitative binding assays to define TER sequence requirements for recognition by the high affinity RNA binding domain and an independent N-terminal RNA interaction domain. The TERT RNA binding domain and N-terminal RNA interaction domain had distinct, nonoverlapping requirements for TER sequence and structure that together accounted for all of the sites of TER contact inferred for full-length TERT. The TER residues important for TERT binding are only a subset of the residues required for catalytic activity. Our findings demonstrate telomerase functional specialization by an elaborate ribonucleoprotein architecture physically separable from the active site.

Animals↗