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PubMed1998

The reverse transcriptase component of the Tetrahymena telomerase ribonucleoprotein complex.

Telomerase is a eukaryotic reverse transcriptase that adds simple sequence repeats to chromosome ends by copying a template sequence within the RNA component of the enzyme. We describe here the identification of a Tetrahymena telomerase protein with reverse transcriptase motifs, p133. This subunit is associated with the previously identified Tetrahymena telomerase RNA and the telomerase proteins p80 and p95 in immunoprecipitation assays. Therefore, all four known Tetrahymena telomerase components are present in a single complex. Expressed in rabbit reticulocyte lysate, recombinant p133 and telomerase RNA alone catalyze a reverse transcriptase activity with some similarities to and some differences from native Tetrahymena telomerase. These experiments suggest a complexity of telomerase structure and function.

Amino Acid Sequence↗

PubMed1989

Virus-like particles associated with reverse transcriptase activity in acute sporadic non-A,non-B hepatitis.

Reverse transcriptase activity was tested in 65 patients with non-A,non-B hepatitis, with positive results in 2 acute sporadic cases with favorable outcome. Virus-like particles were observed in ultra-thin sections of successive serum samples from one of the reverse transcriptase activity-positive patients by electron microscopy. These results suggest that some non-A,non-B hepatitis types could be related to a virus-like agent associated with a reverse transcriptase activity.

Adult↗

PubMed1994

Subunit-selective mutagenesis of Glu-89 residue in human immunodeficiency virus reverse transcriptase. Contribution of p66 and p51 subunits to nucleoside analog sensitivity, divalent cation preference, and steady state kinetic properties.

The E89G alteration in the human immunodeficiency virus type 1 reverse transcriptase has been shown to confer resistance to nucleoside analogs and a loss of magnesium cation preference (Prasad, V.R., Lowy, I., De Los Santos, T., Chiang, L., and Goff, S.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11363-11367. The wild type reverse transcriptase heterodimer, chimeric reverse transcriptases that contain the E89G alteration in one of the subunits (p66wt/p51m and p66m/p51wt), and the mutant enzyme (p66m/p51m) were prepared. Analysis of steady state kinetic parameters showed that the mutant enzyme (p66m/p51m) displayed a higher Vmax, a higher Km for 2'-deoxythymidine triphosphate, and a higher Ki for 2',3'-dideoxythymidine triphosphate than the wild type enzyme. The increased Km and Ki values were observed only when a heterodimer contained the alteration in the p66 subunit. Tests for divalent cation requirement showed that only the dimers containing the wild type p66 (p66wt/p51wt and p66wt/p51m) displayed a preference for magnesium. Our results indicate that p66 plays a dominant role in deoxynucleotide triphosphate substrate recognition (Km), nucleoside analog sensitivity (Ki), and magnesium preference. However, the increased Vmax displayed by the mutant enzyme (p66m/p51m) appeared to be determined by both of the subunits.

Base Sequence↗

PubMed2001

Impact of clinical reverse transcriptase sequences on the replication capacity of HIV-1 drug-resistant mutants.

We have shown that the HIV-1 laboratory strain NL4-3 that contains P236L [a reverse transcriptase mutation conferring resistance to the nonnucleoside reverse transcriptase inhibitor (NRTI) delavirdine] replicates more slowly than wild-type NL4-3. Other NNRTI-resistance mutations, such as K103N and Y181C, do not reduce the replication capacity of NL4-3 as much as P236L and develop more frequently in HIV-1 isolates from patients failing delavirdine. However, a minority of patients on delavirdine therapy still have isolates with P236L. We postulated that reverse transcriptase (RT) sequences from these patient isolates contain other mutations that compensate for the adverse effect of P236L. To test this hypothesis, we created 15 chimeric NL4-3 isolates that contained delavirdine-resistant RT sequences derived from eight patient isolates and characterized their replication kinetics. Nine of 10 patient-derived clones containing P236L replicated as slowly as NL4-3 with P236L. In contrast, three of five clones that did not have P236L (but had either K103N or Y181C) replicated significantly better than NL4-3 with P236L. Thus, the majority of patients who acquire P236L during delavirdine therapy do not have RT mutations that compensate for the replication defect conferred by P236L. We hypothesize that HIV-1 isolates with P236L may have a compensatory mutation outside RT. Alternatively, variants of HIV-1 with reduced replication fitness may be selected during antiretroviral therapy, suggesting that stochastic events rather than viral replication fitness may determine which drug-resistant mutants emerge early during antiretroviral failure. In some isolates, it appears that the background RT sequence can contribute significantly to the replication fitness of drug-resistant HIV-1 variants.

Anti-HIV Agents↗

PubMed1995

Effect of dimethyl sulfoxide on reverse transcriptase activity.

At low concentrations, dimethyl sulfoxide (DMSO) stimulated the avian myeloblastosis virus reverse transcriptase activity. About 40% stimulation was obtained in the presence of 5% (v/v) DMSO, using activated DNA and polyriboadenylic acid (poly(rA)) as templates, and Mg2+ as divalent cation. A similar stimulation by DMSO was observed with Mn2+ for the poly(rA)-dependent reverse transcriptase activity. DMSO at concentrations higher than 15% inhibited the reverse transcriptase reactions, independent of the template-primers used. An exception was detected with the 2'-fluoro analog of poly(rA) as template, where an activation of 100% was found in the presence of 20% DMSO. The stimulation caused by DMSO could be due to a reduction of the apparent Km value for poly(rA) from 9.1 to 3.3 micrograms/ml.

Avian Myeloblastosis Virus↗

PubMed2005

Sensitive phenotypic detection of minor drug-resistant human immunodeficiency virus type 1 reverse transcriptase variants.

Detection of drug-resistant variants is important for the clinical management of human immunodeficiency virus type 1 (HIV-1) infection and for studies on the evolution of drug resistance. Here we show that hybrid elements composed of the Saccharomyces cerevisiae retrotransposon Ty1 and the reverse transcriptase (RT) of HIV-1 are useful tools for detecting, monitoring, and isolating drug-resistant reverse transcriptases. This sensitive phenotypic assay is able to detect nonnucleoside reverse transcriptase inhibitor-resistant RT domains derived from mixtures of infectious molecular clones of HIV-1 in plasma and from clinical samples when the variants comprise as little as 0.3 to 1% of the virus population. Our assay can characterize the activities and drug susceptibilities of both known and novel reverse transcriptase variants and should prove useful in studies of the evolution and clinical significance of minor drug-resistant viral variants.

Drug Resistance, Viral↗

PubMed1979

Recognition of structure of 5-halogenated derivatives of ara-UTP by DNA polymerase gamma and reverse transcriptase.

This report deals with the test of a series of 5-halogenated derivatives of ara-UTP for the inhibition of DNA polymerase gamma and viral reverse transcriptase. The compounds newly synthesized and tested were; ara5-FUTP, ara5-C1UTP, ara5-BrUTP and ara5-IUTP. The results were: 1) All these compounds were inhibitory to DNA polymerase gamma and reverse transcriptase. The mode of inhibitions was, in all cases, competitive against dTTP. 2) Ki values for these inhibitors with DNA polymerase gamma were lower than those with reverse transcriptase. 3) Ara5-ClUTP was less inhibitory to reverse transcriptase than other derivatives.

Animals↗

PubMed1987

Detection of human immunodeficiency virus by reverse transcriptase assay, antigen capture assay, and radioimmunoassay.

The reverse transcriptase assay, antigen capture assay, and radioimmunoassay were compared for the detection of human immunodeficiency virus (HIV) in culture fluids of virus-infected lymphocytes. The reverse transcriptase assay and the antigen capture assay were compared for 962 samples, and the two tests displayed comparable sensitivities (98.5% agreement) in detecting HIV antigen. In addition, these two tests displayed similar sensitivities when examined for the kinetics of HIV appearance following in vitro infection of normal lymphocytes. We also found the antigen capture assay to be as sensitive as the radioimmunoassay in detecting HIV antigen in culture fluids. Furthermore, all three tests were found to be reasonably concordant when applied simultaneously to the detection of HIV antigen in cultures. The antigen capture assay, however, is relatively fast, can handle a large number of samples, does not require radioactive material, and is less expensive than the other two tests. Therefore, the antigen capture assay appears to be the method of choice for the detection of HIV in routine virus culturing.

Antigens, Viral↗

PubMed1990

Reverse transcriptase from Escherichia coli exists as a complex with msDNA and is able to synthesize double-stranded DNA.

Reverse transcriptase required for the synthesis of msDNA.Ec67 in an Escherichia coli strain was purified as a large molecular weight complex with msDNA. The complex sedimented in a glycerol gradient at an s value greater than 19. The predominant protein species co-purifying with reverse transcriptase activity in the complex had a molecular weight estimated at 65,000 which is close to the expected size of 67,227 for the Ec67-reverse transcriptase. In addition, the large complex also contained msDNA.Ec67. The purified complex was able to synthesize cDNA using 5 S rRNA as a template (annealed to a synthetic DNA primer), and a double-stranded DNA using a synthetic DNA template (annealed to a synthetic DNA primer). When msDNA.Ec67 was used as a natural template:primer, the purified complex produced two major products: a 103-base single-stranded DNA by extending the 3' end of msDNA using msdRNA as a template, and a 60-base double-stranded DNA product resulting from the converse reaction in which the 3' end of msdRNA is extended using msDNA as a template. The results suggest that bacterial reverse transcriptase is capable of producing single-stranded cDNA and possibly double-stranded DNA as well. Possible implications of these findings on the biology of the msDNA-retron system are discussed.

Base Sequence↗

PubMed1995

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.

The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.

Antiviral Agents↗

PubMed1996

Quinoxapeptins: novel chromodepsipeptide inhibitors of HIV-1 and HIV-2 reverse transcriptase. I. The producing organism and biological activity.

Quinoxapeptin A and B are novel chromodepsipeptides which were isolated from a nocardioform actinomycete with indeterminant morphology. Quinoxapeptins A and B are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase and almost equally active against two single mutants forms as well as a double mutant form of HIV-1 reverse transcriptase. Quinoxapeptin A and B are specific inhibitors of HIV-1 and HIV-2 reverse transcriptase because they did not inhibit human DNA polymerase alpha, beta, gamma and delta. Quinoxapeptin A and B are structurally similar to luzopeptin A which was also active against HIV-1 and HIV-2 reverse transcriptase.

Actinomycetales↗

PubMed2003

Inhibition of human immunodeficiency virus reverse transcriptase by synadenol triphosphate and its E-isomer.

Triphosphate 1c is a potent competitive inhibitor of wild-type HIV-1 reverse transcriptase with K(i) close to ddATP. The E-isomer 2c is about 30-times weaker. Triphosphates 1c and 2c interact with the same active site of reverse transcriptase as ddATP. The extent of inhibition of two mutant forms of reverse transcriptase (RT), RT(M184V) and RT(M184I), with triphosphate 1c was about 5 and 8 times lower than that of wild-type RT(wt).

Anti-HIV Agents↗

PubMed2002

Inhibitory effects of antifungal proteins on human immunodeficiency virus type 1 reverse transcriptase, protease and integrase.

A variety of antifungal proteins were isolated from seeds of leguminous plants including French bean, cowpea, field bean, mung bean, peanut and red kidney bean. They were assayed for ability to inhibit human immunodeficiency virus type I (HIV-1) reverse transcriptase, protease and integrase, enzymes essential to the life cycle of HIV-1 . It was found that the cowpea beta-antifungal protein had a high potency in inhibiting HIV-1 protease and HIV-1 integrase. Cowpea alpha-antifungal protein was potent in inhibiting HIV-1 reverse transcriptase and HIV-1 integrase. Peanut antifungal protein was characterized by a high inhibitory activity against HIV-1 integrase and an intermediate potency in inhibiting HIV- I reverse transcriptase and HIV- I protease. French bean thaumatin-like protein expressed low HIV- I protease inhibitory activity and red kidney bean lectin inhibited HIV- I integrase by only a very small extent. Antifungal proteins from the field bean and mung bean had an intermediate potency in inhibitory HIV-1 protease and integrase. However, mung bean antifungal protein was not capable of inhibiting HIV-1 reverse transcriptase. The results indicate that nearly all leguminous antifungal proteins examined were able to inhibit HIV-1 reverse transcriptase, protease and integrase to some extent.

Amino Acid Sequence↗

PubMed2006

Phenotypic drug resistance patterns in subtype A HIV-1 clones with nonnucleoside reverse transcriptase resistance mutations.

We analyzed the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI) susceptibility of 29 subtype A HIV-1 clones isolated from 10 Ugandan women after single-dose nevirapine (NVP) administration. Six clones had no NNRTI resistance-associated mutations ("wild type"), eight had K103N, nine had Y181C, five had G190A, and one had Y181S. Three clones displayed unexpected phenotypic drug susceptibility/resistance based on their RT genotypes. One wild-type clone had reduced susceptibility to NVP, delavirdine (DLV), and efavirenz (EFV), one clone with K103N was susceptible to all three NNRTIs, and one clone with G190A had extreme hypersusceptibility to DLV. Three unusual HIV-1 RT amino acid substitutions may have contributed to the unexpected phenotypes of the clones: I31T, N136S, and N265D. These polymorphisms were rarely detected among 47,900 HIV-1 genotypes from clinical samples of predominantly United States origin. Further studies are needed to define the genetic correlates of antiretroviral drug resistance in nonsubtype B HIV-1.

Alkynes↗

PubMed2001

Alternatively spliced variant deleting exons 7 and 8 of the human telomerase reverse transcriptase gene is dominantly expressed in the uterus.

The expression level of the human telomerase reverse transcriptase (hTERT) is a rate-limiting determinant of telomerase activity. Several alternatively spliced variants of hTERT transcript are currently known. We have studied the expression of the splicing variants arising in the transcript encoding the reverse transcriptase domain, and have compared this to the telomerase activity in 27 endometria, 14 myometria and 18 endometrial carcinomas. Telomerase activity and the full-length hTERT transcript were observed in endometrial samples from the proliferative and early secretory phases, but not in those from the late secretory phase. Steady-state expression of the hTERT splicing variant entirely lacking exon 7 and exon 8 was observed in the endometria throughout the menstrual cycle. In the analysed myometria, this type of splicing variant was the most commonly detected, and telomerase activity occurred in only three samples. In both endometria and myometria, the expression of the full-length transcript correlated well with the telomerase activity. In each of the endometrial carcinomas, telomerase activity was detected and the full-length transcript was found together with varying combinations of deletion splicing variants. These results suggest that regulation of splicing in the transcript encoding the hTERT reverse transcriptase domain is associated with telomerase activation in uterine tissues.

Adult↗

PubMed2001

International perspectives on antiretroviral resistance. Nonnucleoside reverse transcriptase inhibitor resistance.

Although understanding of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance is less clearly established than that of other classes of antiretroviral drugs, certain facts have been established. The treatment-associated genetic mutation profiles of the available NNRTIs have been mapped, and resistance has been found to develop rapidly after initiation of NNRTI therapy. Despite the chemical diversity of the NNRTIs, cross-resistance among agents of this class is nearly universal. Although the viral replicative capacity ("fitness") of NNRTI-induced viral variants has not been extensively studied, available data suggest that NNRTI-selected mutations confer little damage to viral fitness, and thus a single point mutation produces a strain that is both resistant and fit. Furthermore, with continued therapy, viral evolution persists, creating species with greater numbers of mutations and higher level phenotypic resistance. Taken together, these facts suggest that continued use of NNRTIs after emergence of resistance will produce variants of complex mutational patterns that limit future treatment options, and, therefore, strong consideration should be given to discontinuing NNRTIs after virologic failure is confirmed. This article describes the scientific literature establishing the efficacy and limitations of NNRTI therapy and attempts to define a role for this class of drug in the long-term treatment of HIV-1 disease.

Alkynes↗

PubMed1999

Initiation of minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The initiation of (-) strand DNA synthesis by HIV-1 reverse transcriptase was examined using a transient kinetic approach and a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. HIV-1 reverse transcriptase (RT) was found to bind with reasonably high affinity to the RNA/RNA substrate (K(d) = 90 nM), although the affinity for DNA/RNA and DNA/DNA substrates is higher (K(d) approximately 5 nM). A pre-steady-state burst of deoxynucleotide incorporation (k(obsd) = 1.0 s(-)(1)) into the RNA duplex was observed followed by a slower steady-state release of the elongated primer-template product (k(ss) = 0.58 s(-)(1)). The observation of a burst provides evidence that the release of the product is most likely the rate-limiting step in the overall kinetic pathway for the enzymatic reaction during a single deoxynucleotide incorporation event. Furthermore, the release of this product was 5-fold faster than that for elongated DNA/RNA and DNA/DNA products. Single-turnover experiments showed that there is a hyperbolic dependence of the rate of deoxynucleotide incorporation on the concentration of dCTP and demonstrated that the maximum rate of dCTP incorporation (k(pol) = 1.4 s(-)(1)) is 33- and 12-fold slower than the values for DNA/RNA and DNA/DNA primer-template substrates, respectively, while the affinity of dCTP (K(d) = 780 microM) for the HIV-1 RT.RNA/RNA complex is 56- and 71-fold weaker than the affinities for HIV-1 RT.DNA/RNA and HIV-1 RT.DNA/DNA complexes, respectively. Consequently, the overall efficiency of dCTP incorporation (k(pol)/K(d)) into the RNA/RNA substrate is approximately 1800- and 800-fold less than that for DNA/RNA and DNA/DNA substrates, respectively. These findings provide evidence which suggests that the HIV-1 RT.RNA/RNA.dCTP ternary complex exists in a significantly different conformation compared to ternary complexes involving DNA/RNA and DNA/DNA substrates. A model summarizing these results is presented, and implications for the molecular mechanism of initiation of (-) strand DNA synthesis by RT are discussed.

Binding Sites↗

PubMed1993

Comprehensive mutant enzyme and viral variant assessment of human immunodeficiency virus type 1 reverse transcriptase resistance to nonnucleoside inhibitors.

The nonnucleoside reverse transcriptase (RT) inhibitors comprise a class of structurally diverse compounds that are functionally related and specific for the human immunodeficiency virus type 1 RT. Viral variants resistant to these compounds arise readily in cell culture and in treated, infected human. Therefore, the eventual clinical usefulness of the nonnucleoside inhibitors will rely on a thorough understanding of the genetic and biochemical bases for resistance. A study was performed to assess the effects of substitutions at each RT amino acid residue that influences the enzyme's susceptibility to the various nonnucleoside compounds. Single substitutions were introduced into both purified enzyme and virus. The resulting patterns of resistance were markedly distinct for each of the tested inhibitors. For instance, a > 50-fold loss of enzyme susceptibility to BI-RG-587 was engendered by any of four individual substitutions, while the same level of relative resistance to the pyridinone derivatives was mediated only by substitution at residue 181. Similarly, substitution at residue 181. Similarly, substitution at residue 106 had a noted effect on virus resistance to BI-RG-587 but not to the pyridinones. The opposite effect was mediated by a substitution at residue 179. Such knowledge of nonucleoside inhibitor resistance profiles may help in understanding the basis for resistant virus selection during clinical studies of these compounds.

Acquired Immunodeficiency Syndrome↗