Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HIV Reverse Transcriptase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed1999

Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the 3TC-resistant mutant (184V) of HIV-1 reverse transcriptase.

A single amino acid substitution from methionine-184 to valine (M184V) of HIV-1 reverse transcriptase (RT) evokes the 1000-fold 3TC (Lamivudine) resistance by the HIV-1 virus observed in the clinic. The M184V mutant HIV-1 RT was studied to assess its catalytic efficiency during single nucleotide incorporation using a transient kinetic approach. The maximum rate of polymerization (k(pol)), binding affinity (K(d)), and incorporation efficiency (k(pol)/K(d)) were determined for incorporating dCTP and 3TC-TP by wild-type and 3TC-resistant HIV-1 RT. The 3TC-resistant HIV-1 RT showed a similar efficiency of incorporation compared with the wild-type enzyme during DNA-dependent DNA polymerization; however, the incorporation efficiency is reduced 3.5-fold during RNA-dependent polymerization. A dramatic 146- and 117-fold decrease in incorporation efficiency was observed for 3TC-MP incorporation by M184V RT for DNA- and RNA-dependent DNA polymerization, respectively, as compared with wild-type HIV-1 RT. While the k(pol) was slower and the K(d) was weaker for 3TC-TP incorporation by the M184V RT, the decrease in the efficiency of incorporation is primarily due to a substantially reduced binding affinity for the 3TC-TP to the enzyme.DNA (or RNA) complex poised for DNA elongation. The fidelity of M184V RT was also examined to evaluate mispair formation since this mutant has been suggested to exhibit a higher level of fidelity. The results of our studies indicate that there is a maximum 2.4-fold increase in fidelity for M184V RT as compared with wild-type HIV-1 RT. Both the wild-type and 3TC-resistant mutant RT showed higher fidelity using an RNA template as contrasted with the corresponding DNA template. This mechanistic information provides insight into our understanding of the molecular mechanism of 3TC-drug resistance and supports suggestions that increased RT fidelity and decreased fitness of the M184V HIV-1 virus may be factors contributing to the strong antiviral effect of AZT-3TC combination therapy.

Amino Acid Substitution↗
PubMed1998

Stable rearrangements of the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase in plasma viruses from patients receiving combination therapy.

OBJECTIVES: To study the genetic rearrangements of HIV-1 reverse transcriptase (RT) in circulating viruses from patients under combination therapy, and to determine the impact of these changes on the virological response to treatment. METHODS: Blood samples were extracted from total RNA and amplified by RT-PCR. The HIV-1 RT and protease genes were sequenced by fluorescent dye terminator cycle sequencing. RESULTS: Specific rearrangements in the RT coding region (between amino acids 66 and 71) were documented in nine patients. This region, which corresponds to a loop between the beta3 and beta4 strands of the fingers subdomain of RT, is involved in the interaction between the enzyme and the template primer. In vitro data with recombinant enzymes have shown the importance of this domain in the processive polymerization of HIV-1 RT. The rearrangements (eight deletions/insertions and one deletion with conservation of the reading frame) did not affect the overall secondary structure of the fingers subdomain, as assessed by the Garnier Osguthorpe Robson prediction method. The changes were generally stable over a follow-up of 10-12 months. With the exception of two cases, most of the patients of this study did not respond efficiently to antiretroviral therapy as assessed by measurements of plasma viraemia. Correspondingly, the RT and protease genes sequenced from these patients displayed numerous resistance-associated mutations. CONCLUSION: Functional and stable rearrangements in the beta3-beta4 hairpin of HIV-1 RT can be found in circulating viruses from patients under combination therapy. These rearrangements may affect the virological response to antiretroviral therapy by increasing the processivity of RT, an enzymatic parameter that reflects the fidelity of the polymerization process.

Amino Acid Sequence↗
PubMed1997

Synthesis and biological activity of novel nonnucleoside inhibitors of HIV-1 reverse transcriptase. 2-Aryl-substituted benzimidazoles.

The development of new nonnucleoside inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) active against the drug-induced mutations in RT continues to be a very important goal of AIDS research. We used a known inhibitor of HIV-1 RT, 1-(2,6-difluorophenyl)-1H,3H-thiazolo[3,4-alpha]benzimidazole (TZB), as the lead structure for drug design with the objective of making more potent inhibitors against both wild-type (WT) and variant RTs. A series of structurally related 1,2-substituted benzimidazoles was synthesized and evaluated for their ability to inhibit in vitro polymerization by HIV-1 WT RT. A structure-activity study was carried out for the series of compounds to determine the optimum groups for substitution of the benzimidazole ring at the N1 and C2 positions. The best inhibitor, 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-4-methylbenzimida zole (35), has an IC50 = 200 nM against HIV-1 WT RT in an in vitro enzyme assay. Cytoprotection assays utilizing HIV-infected MT-4 cells revealed that 35 had strong antiviral activity (EC50 = 440 nM) against wild-type virus while retaining broad activity against many clinically observed HIV-1 strains resistant to nonnucleoside inhibitors. Overall, the activity of 35 against wild-type and resistant strains with amino acid substitution in RT is 4-fold or greater than that of TZB and is comparable to that of other nonnucleoside inhibitors currently undergoing clinical trials, most of which do not have the capacity to inhibit the variant forms of the enzyme.

Anti-HIV Agents↗
PubMed2002

Synthesis and anti-HIV activity of [d4U]-[trovirdine analogue] and [d4T]-[trovirdine analogue] heterodimers as inhibitors of HIV-1 reverse transcriptase.

A series of eleven heterodimers containing both a nucleoside analogue (d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue) were synthesized and evaluated for their ability to inhibit HIV replication. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a-e) and (N-3)d4T-Trovirdine conjugates (10a-f) were found to be inactive suggesting that the two individual inhibitor compounds do not bind simultaneously in their respective sites.

Anti-HIV Agents↗
PubMed2001

Identification of a putative binding site for [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)thymine (TSAO) derivatives at the p51-p66 interface of HIV-1 reverse transcriptase.

A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.

Amino Acid Substitution↗
PubMed1994

Effect of template secondary structure on the inhibition of HIV-1 reverse transcriptase by a pyridinone non-nucleoside inhibitor.

The importance of RNA secondary structure on HIV-1 reverse transcriptase catalyzed polymerization and on the potency of the pyridin-2-one inhibitor 3-(4,7-dichlorobenzoxazol-2-ylmethylamino)-5-ethyl-6-meth ylpyridin-2(1H)-one, L-697,661, were investigated by employing heteromeric primer-template systems. Our data revealed that a stem-loop hairpin secondary structure in the RNA template could lead to strong hindrance of reverse transcription in the reaction catalyzed by HIV-1 reverse transcriptase resulting in the build up of intermediate-length (pause) polymerization products. The presence of L-697,661 greatly enhanced the accumulation of the pause products suggesting that the rate of enzyme translocation from the pause product might be more potently inhibited than polymerization up to the pause site. Model experiments using a synthetic RNA template containing a stem-loop hairpin revealed that the inhibitory potency of L-697, 661 increased 2-fold upon polymerization to within four bases of the secondary structure. Inhibitor potency was enhanced over 6-fold when primer-extension proceeded through the duplex region of the stem-loop.

Animals↗
PubMed1994

Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction.

We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication. Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription. The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template. Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product. Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3). When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching. Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching. Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching.

DNA Primers↗
PubMed2004

Vif is an auxiliary factor of the HIV-1 reverse transcriptase and facilitates abasic site bypass.

The HIV-1 accessory protein Vif was found to modulate the RNA- and DNA-dependent DNA synthesis activity of the viral RT (reverse transcriptase) in two ways: (i) it stimulated the binding of the viral RT to the primer by increasing the association rate kcat/K(m) and by decreasing the thermodynamic barrier DeltaH([ES]) for complex formation, and (ii) it increased the polymerization rate of HIV-1 RT. A Vif mutant lacking the final 56 amino acids at the C-terminus failed to stimulate the viral RT. On the other hand, another Vif mutant lacking the first 43 amino acids at the N-terminus, which are involved in RNA binding and interaction with the viral protease, was able to stimulate RT activity. In addition, Vif was found to promote the bypass of an abasic site by HIV-1 RT.

DNA Primers↗
PubMed1994

Synthesis of a series of 4-(arylethynyl)-6-chloro-4-cyclopropyl-3,4-dihydroquinazolin-2(1H)-ones as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

As part of an ongoing effort to prepare novel non-nucleoside inhibitors of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT), a series of 4-(arylethynyl)-6-chloro-4-cyclopropyl-3,4-dihydroquinazolin -2(1H)-ones 4aa-l has been prepared. Target compounds 4a-e were synthesized via addition of various 1-lithio-2-(aryl)alkyne nucleophiles to a 1-protected-4-cyclopropylquinazolin-2(1H)-one (7), followed by deprotection. The 3-methyl compound 4aa was prepared in an analogous manner, with the 3-alkylation performed prior to deprotection. Alternatively, the target compounds 4f-l were prepared by addition of 1-lithio-2-(trimethylsilyl)acetylene to 7, followed by deprotection and subsequent palladium-catalyzed coupling with various aryl halides. By incorporating an aryl group onto the end of the 4-acetylene functionality, the requirement for a metabolically labile 3-methyl group on the dihydroquinazolinone nucleus has been eliminated. A number of the target compounds were shown to be potent inhibitors of HIV-1 RT. Compound 4a, which had exhibited the most favorable overall biological profile, was resolved via a four-step procedure to provide the enantiomers 13a and 13b. Compound 13a having the (-)-4(S) configuration was shown to be the active enantiomer and was selected as a candidate for further investigation.

Cells, Cultured↗
PubMed2006

Structural insights into mechanisms of non-nucleoside drug resistance for HIV-1 reverse transcriptases mutated at codons 101 or 138.

Lys101Glu is a drug resistance mutation in reverse transcriptase clinically observed in HIV-1 from infected patients treated with the non-nucleoside inhibitor (NNRTI) drugs nevirapine and efavirenz. In contrast to many NNRTI resistance mutations, Lys101(p66 subunit) is positioned at the surface of the NNRTI pocket where it interacts across the reverse transcriptase (RT) subunit interface with Glu138(p51 subunit). However, nevirapine contacts Lys101 and Glu138 only indirectly, via water molecules, thus the structural basis of drug resistance induced by Lys101Glu is unclear. We have determined crystal structures of RT(Glu138Lys) and RT(Lys101Glu) in complexes with nevirapine to 2.5 A, allowing the determination of water structure within the NNRTI-binding pocket, essential for an understanding of nevirapine binding. Both RT(Glu138Lys) and RT(Lys101Glu) have remarkably similar protein conformations to wild-type RT, except for significant movement of the mutated side-chains away from the NNRTI pocket induced by charge inversion. There are also small shifts in the position of nevirapine for both mutant structures which may influence ring stacking interactions with Tyr181. However, the reduction in hydrogen bonds in the drug-water-side-chain network resulting from the mutated side-chain movement appears to be the most significant contribution to nevirapine resistance for RT(Lys101Glu). The movement of Glu101 away from the NNRTI pocket can also explain the resistance of RT(Lys101Glu) to efavirenz but in this case is due to a loss of side-chain contacts with the drug. RT(Lys101Glu) is thus a distinctive NNRTI resistance mutant in that it can give rise to both direct and indirect mechanisms of drug resistance, which are inhibitor-dependent.

Anti-HIV Agents↗
PubMed2000

HIV-1 reverse transcriptase (RT) genotype and susceptibility to RT inhibitors during abacavir monotherapy and combination therapy.

OBJECTIVE: To examine changes in HIV-1 susceptibility (genotype and phenotype) during an initial abacavir monotherapy phase followed by the addition of zidovudine and lamivudine. DESIGN: Sixty HIV-1 infected, antiretroviral therapy-naive subjects were randomized to receive 100, 300 or 600 mg abacavir twice daily. Subjects completing 24 weeks of randomized therapy or meeting a protocol defined switch criterion could switch to open label abacavir/zidovudine/lamivudine. METHODS: Plasma HIV-1 reverse transcriptase was genotyped at baseline, week 12, and at the last time point on ABC monotherapy. Drug susceptibility was analysed at baseline and on subsequent samples with sufficient HIV-1 RNA levels using the recombinant virus assay. Virological responses (week 24) were correlated to week 24 genotypes. RESULTS: Mutant viruses were not detected before week 12 with the exception of one subject. At the latest time point on abacavir monotherapy (range, weeks 6-48), 21 out of 43 subjects harboured virus with resistance conferring mutations including single, double and triple combinations of K65R, L74V, Y115F and M184V. The most common mutational pattern was L74V + M184V (11/21 cases). Twenty of the 21 subjects with isolates containing abacavir-associated mutations reached week 48, and upon addition of lamivudine/zidovudiine, 16 out of 20 (80%) had week 48 plasma HIV-1 -RNA below 400 copies/ml. At week 48, 16 out of 46 genotypes were obtained; one of these was wild-type; 15 contained M184V either alone, in combination with K65R and/or L74V and/or Y115F or with thymidine analogue-associated mutations. Week 48 viral load levels for these 15 subjects was low (median 3.43 log10 copies/ml or -1.99 log10 copies reduction from baseline). Genotype correlated well with phenotypic resistance to ABC; four samples with three abacavir-associated mutations had high level abacavir resistance (> 8-fold) and six samples with two or three mutations showed intermediate (4-8-fold) resistance. All samples with single mutations retained full ABC susceptibility. CONCLUSIONS: Resistance conferring mutations to abacavir were relatively slow to develop during the monotherapy phase, and did not preclude durable efficacy of abacavir/lamivudine/zidovudine up to 48 weeks.

Cohort Studies↗
PubMed2003

Nevirapine resistance mutation at codon 181 of the HIV-1 reverse transcriptase confers stavudine resistance by increasing nucleotide substrate discrimination and phosphorolytic activity.

Recombinant HIV-1 reverse transcriptase (RT) carrying non-nucleoside inhibitors (NNRTIs) resistance mutation at codon 181 showed reduced incorporation and high efficiency of phosphorolytic removal of stavudine, a nucleoside RT inhibitor. These results reveal a new mechanism for cross-resistance between different classes of HIV-1 RT inhibitors.

Codon↗
PubMed2004

QSAR modeling of HIV-1 reverse transcriptase inhibitor 2-amino-6-arylsulfonylbenzonitriles and congeners using molecular connectivity and E-state parameters.

Anti-HIV-1 activity (assayed in MT-4 cell line) and HIV-1 reverse transcriptase (RT) binding affinity of 2-amino-6-arylsulfonylbenzonitriles and their thio and sulfinyl congeners (Chan et al., J. Med. Chem., 2001, 44, 1866) have been modeled using E-state index along with molecular connectivity and indicator parameters in an attempt to explore the different fragments of the molecules contributing significantly to the activities. Stepwise multiple regression procedure was adopted to develop the topological models. The models generated were of acceptable statistical quality and predictive potential. The results show that for both the response variables, first order fragmental valence molecular connectivity of the meta substituents of the aryl ring plays a significant role: second meta substituents show supraadditive action on the activities probably due to enhanced binding (presumably through dispersion interaction) of the ligand with the binding site. Again, presence of sulfone moiety contributes significantly to the activities. Further, presence of meta-trifluoromethyl group at the aryl ring is detrimental for both the activity parameters. Additionally, the anti-HIV-1 model shows specific contributions of the E-state values of different atoms and positive contribution of the ortho-methoxy group present on the aryl ring.

Amination↗
PubMed1993

Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label.

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.

Affinity Labels↗
PubMed1995

Conformational stability of dimeric HIV-1 and HIV-2 reverse transcriptases.

The dissociation of dimeric reverse transcriptase (RT) of the human immunodeficiency virus (HIV) types 1 and 2 has been investigated using acetonitrile as a dissociating agent. The equilibrium transitions were monitored by combining different approaches (fluorescence spectroscopy, polymerase activity assay, and size-exclusion HPLC). The dissociation of RT induced a complete loss of polymerase activity and a 25% increase of the intrinsic fluorescence. It is fully reversible, and the midpoints of the equilibrium transition curves are dependent on the concentration of the enzyme used, suggesting a two-state transition model for the dissociation of RT in which dimers are in equilibrium with folded monomers. For both RTs, the heterodimeric form is more stable against dissociating agents and different pH than the corresponding homodimeric form. Moreover, heterodimeric HIV-2 RT exhibits a higher stability than HIV-1 RT, with a free energy of dissociation of 12.1 kcal/mol at pH 6.5 and 25 degrees C, instead of 10 kcal/mol for HIV-1 RT. The binding of a primer/template induces a marked conformational change in both RTs, shown by the lower accessibility of the tryptophans to quenchers and the increase in tryptophan heterogeneity, and stabilized the dimeric form of both RTs (10-100-fold). The central role of hydrophobic interactions in dimer formation has been revealed by the 30% increase of exposure of the tryptophan cluster to quenchers upon dissociation of RT and the binding of 4 equiv of 1-anilino-8-naphthalenesulfonate to the dissociated enzymes.

Acetonitriles↗
PubMed1989

Presence of HIV-1 reverse transcriptase inhibitory antibodies in the sera of some HTLV-1 carriers and absence of neutralizing antibodies against HIV-1 in the sera of HTLV-1 carriers.

We have detected inhibitory antibody against HIV-1 reverse transcriptase (RT) in the sera of HTLV-1 carriers, although the lack of immunological cross reaction between HIV-RT and other mammalian retroviruses, including HTLV-1, has been reported elsewhere (1). Sera from 72 HTLV-1 carriers in the southern part of Kyushu in Japan and 61 controls from the same district were studied and controlled for age and sex. One of the 72 HTLV-1 carriers was HTLV-1 antibody-positive, while the others were HIV-1 antibody-negative by ELISA and Western blot method. Sera from two HTLV-1 carriers and one hemophiliac with combined HIV-1 and HTLV-1 infections were found to inhibit HIV-1 RT activity. The inhibition ratios were 72, 65 and 72%, respectively. Protein A effluents of inhibitory sera retained no inhibitory capacity. These results might suggest that HTLV-1 and HIV-1 reverse transcriptase may share common or similar epitopes and that antibodies raised to the HTLV-1 RT react with that of HIV-1 RT in an inhibitory mode. However, the RT inhibitory antibody-positive sera had no neutralizing capacity against HIV-1 infection, supporting the idea that HIV-1 RT inhibitory antibody may not contribute to neutralization against HIV-1. The results also suggest that previous exposure to HTLV-1 does not contribute to neutralizing antibodies against HIV-1 in HIV-1 infected individuals.

Blotting, Western↗