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At least 289 records · Page 16Linked to original sources
PubMed1996

Concomitant combination therapy for HIV infection preferable over sequential therapy with 3TC and non-nucleoside reverse transcriptase inhibitors.

Exposure to 3TC of HIV-1 mutant strains containing non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific mutations in their reverse transcriptase (RT) easily selected for double-mutant viruses that had acquired the characteristic 184-Ile mutation in their RT in addition to the NNRTI-specific mutations. Conversely, exposure of 3TC-resistant 184-Val mutant HIV-1 strains to nine different NNRTIs resulted in the rapid emergence of NNRTI-resistant virus strains at a time that was not more delayed than when wild-type HIV-1(IIIB) was exposed to the same compounds. The RTs of these resistant virus strains had acquired the NNRTI-characteristic mutations in addition to the preexisting 184-Val mutation. Surprisingly, when the 184-Ile mutant HIV-1 was exposed to a variety of NNRTIs, the 188-His mutation invariably occurred concomitantly with the 184-Ile mutation in the HIV-1 RT. Breakthrough of this double-mutant virus was markedly accelerated as compared with the mutant virus selected from the wild-type or 184-Val mutant HIV-1 strain. The double (184-Ile + 188-His) mutant virus showed a much more profound resistance profile against the NNRTIs than the 188-His HIV-1 mutant. In contrast with the sequential chemotherapy, concomitant combination treatment of HIV-1-infected cells with 3TC and a variety of NNRTIs resulted in a dramatic delay of virus breakthrough and resistance development.

Base Sequence↗
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↗
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↗
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↗
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↗
PubMed1992

Domain structure of the human immunodeficiency virus reverse transcriptase.

The spatial arrangement of subunits p51 and p66 of the HIV-1 reverse transcriptase and the position of the RNase H containing domain, p15, have been determined by means of neutron small-angle scattering. The reverse transcriptase (p66/p51) is a flat molecule, which can be approximated by an ellipsoid with the half axes of 5.2 nm, 4.8 nm and 1.4 nm. The two subunits p51 and p66 having a centre-to-centre distance of 3.3 +/- 0.3 nm are attached at their flat sides, slightly shifted sideways. The p15 domain is located at the long axis of the ellipsoidal reverse transcriptase having a distance of 5.0 +/- 0.5 nm to the centre of the p51d domain, which is part of the p66 subunit, and a distance of 5.3 +/- 1.2 nm to the centre of the neighbouring p51s subunit.

Chromatography, Affinity↗
PubMed1997

HIV-1 drug susceptibilities and reverse transcriptase mutations in patients receiving combination therapy with didanosine and delavirdine.

Previous studies have shown that the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase mutation Y181C, which confers high-level resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs), develops rarely during therapy with NNRTIs plus zidovudine. To determine whether didanosine (ddI) is also effective in preventing the emergence of Y181C, we analyzed delavirdine (DLV) susceptibilties and reverse transcriptase sequences of isolates obtained from patients enrolled in a pharmacokinetic study of DLV and ddI. Nine NNRTI-naive patients were evaluated. Seven received DLV/ddI and two received DLV/ddI/zidovudine. Median durations of prior zidovudine and ddI were 26 and 15 months, respectively. Isolates from eight of nine patients had a mutation(s) associated with nucleoside resistance at entry. After treatment with DLV and ddI alone, isolates from five of seven patients developed Y181C, four in combination with K103N. Thus, in this group of nucleoside-experienced patients, combination therapy with DLV/ddI did not prevent the emergence of Y181C.

Adult↗
PubMed1993

Biochemical analysis of human immunodeficiency virus-1 reverse transcriptase containing a mutation at position lysine 263.

Site-directed mutagenesis has been used to assess the importance of lysine 263 in substrate binding of human immunodeficiency virus-1 (HIV-1) reverse transcriptase. Previous studies have indicated that lysine 263 functions in the binding of 2'-deoxynucleoside 5'-triphosphate (dNTP) substrates (Basu, A., Tirumalai, R. S., and Modak, M. J. (1989) J. Biol. Chem. 264, 8746-8752). We studied this interaction directly by using site-specific mutagenesis to change lysine 263 to a serine. Highly purified mutant enzyme K263S bound natural dNTP substrates and primed polynucleic acid substrates with equal affinity when compared to the wild type reverse transcriptase. No difference was observed in the binding of 3'-azido-2',3'-dideoxythymidine 5'-triphosphate to the mutant reverse transcriptase on the basis of Km and Ki determinations. The serine substitution had no effect on RNase H activity. These results indicate that lysine 263 is not essential in the binding of substrates to HIV-1 reverse transcriptase.

Base Sequence↗
PubMed1991

Reconstitution in vitro of RNase H activity by using purified N-terminal and C-terminal domains of human immunodeficiency virus type 1 reverse transcriptase.

Two constituent protein domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase were expressed separately and purified to homogeneity. The N-terminal domain (p51) behaves as a monomeric protein exhibiting salt-sensitive DNA polymerase activity. The C-terminal domain (p15) on its own has no detectable RNase H activity. However, the combination of both isolated p51 and p15 in vitro leads to reconstitution of RNase H activity on a defined substrate. These results demonstrate that domains of HIV-1 reverse transcriptase are functionally interdependent to a much higher degree than in the case of reverse transcriptase from Moloney murine leukemia virus.

Base Sequence↗
PubMed1997

The role of genotypic heterogeneity in wild type virus populations on the selection of nonnucleoside reverse transcriptase inhibitor-resistant viruses.

Virus populations were selected in cell culture using two widely used protocols in order to evaluate the role of selection methodology on the genotype and phenotype of nonnucleoside reverse transcriptase inhibitor resistant viruses. Selection was performed by serial passage of virus in the presence of gradually increasing concentrations of antiviral compound or passage in the presence of a constant high concentration of compound. Using the CEM-SS cell line, the IIIB strain of HIV-1, and identical nonnucleoside reverse transcriptase inhibitors, resistant viruses were obtained and their phenotypic and genotypic properties were defined. Resistant virus populations containing the Y181C amino acid change in the reverse transcriptase were predominantly selected with each of the tested compounds. Several of the compounds selected secondary amino acid changes using both methods. A comparison of the resistant viruses selected in our laboratory using each of the two protocols with viruses reported by a second laboratory employing one of the two methods suggests that genotypic differences in the selected virus isolates may most likely result from the variation in the genetic composition of the respective wild type virus pools, rather than the specific selection methodology employed. These results imply that HIV may select a wide variety of amino acid changes to avoid the inhibitory effects of the nonnucleoside reverse transcriptase inhibitors and the selection of compounds for clinical use in combination with agents possessing non-overlapping resistance phenotypes will require evaluation of the agents against virus isolates possessing each of the mutations known to confer drug resistance.

Anti-HIV Agents↗