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PubMed2006

QSAR for non-nucleoside inhibitors of HIV-1 reverse transcriptase.

By means of QSAR algorithms we model the potency pIC(90) [mM] of 154 non-nucleoside reverse transcriptase inhibitors (NNRTI) of the wild-type HIV-1 virus, considered as the second generation analogues of Efavirenz. In addition, 56 inhibitors of the K-103N viral mutant form are also investigated. A pool of 1494 theoretical molecular descriptors provided mainly by the Dragon 5 software is explored by several methods of variable selection: forward stepwise regression, the replacement method, and the genetic algorithm approach. The optimal models found include up to seven parameters: R = 0.7991, R(l-20%-o) = 0.7233 for the case of wild-type, and R = 0.9261, R(l-5%-o) = 0.8802 for the K-103N mutation.

Alkynes↗
PubMed1999

Cell cycle control of reverse transcriptase activity for the yeast retrotransposon Ty3.

Retroviruses and retrotransposons depend on their host cells to complete their replication cycles. In many cases, the viral step of reverse transcription is blocked when host cells are arrested in their cell cycle and this block is only released when the host cell resumes division. It has previously been shown that a retrotransposon found in Saccharomyces cerevisiae, Ty3, is unable to produce full-length double-stranded DNA, the product of reverse transcription, when the host cells are arrested in G1. In this study we show that, although Ty3 viruslike particles are produced at equivalent levels in arrested and nonarrested cells, the reverse transcriptase enzyme is inactive in arrested cells. The enzyme activity is restored when the arrested cells are permitted to resume cell division. These data suggest that a host factor regulates the activity of Ty3 reverse transcriptase in the cell cycle, which represents a novel cellular control of retroelements.

G1 Phase↗
PubMed2004

APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase.

In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G --> A hypermutants derived from a Delta(vif) virus. By using an RNaseH- form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication.

APOBEC-3G Deaminase↗
PubMed1992

Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates.

We have examined the parameters that determine the length and distribution of products synthesized processively by the human immunodeficiency virus reverse transcriptase (HIV-RT). On native or homopolymer templates, the overall length distribution of processively synthesized products is increased by increased temperature or deoxynucleoside triphosphate concentration, or decreased ionic strength. Specific terminations of processive synthesis on either native DNA or RNA templates occur most frequently at positions where the reverse transcriptase (RT) pauses during synthesis. These sites correlate with the template sequence 3'-(A/U)(A/U)(G/C)-5', particularly when this sequence is predicted to be base paired with another region of the template in a secondary structure. Many positions of termination are in similar positions on DNA or RNA templates. Notable exceptions are runs of A residues, which promote termination on DNA but not RNA templates. Termination intensities vary when different RTs are used demonstrating an influence of RT structure.

Base Sequence↗
PubMed2000

Screening of selected plant extracts for in vitro inhibitory activity on HIV-1 reverse transcriptase (HIV-1 RT).

Methanolic-aqueous extracts of 70 plants were investigated for their ability to inhibit HIV-1 reverse transcriptase activity in vitro. Two thirds of the extracts screened showed more than 50% inhibition. Two extracts inhibited the enzyme completely while four exhibited more than 90% inhibition. Tannins as nonspecific HIV-1 RT inhibitors were detected and removed from the extracts. The IC50 values of the most potent extracts after the removal of tannins for the HIV-1 RT inhibition are as follows: Sambucus racemosa 0.017 mg/ml and Geranium phaeum 0.067 mg/ml. Daunomycine was chosen as a standard substance in the non-radioactive immuno assay used for screening. As a result from the future isolation and characterization of these compounds, new leading structures are expectable.

Enzyme-Linked Immunosorbent Assay↗
PubMed2004

Effects of the G190A substitution of HIV reverse transcriptase on phenotypic susceptibility of patient isolates to delavirdine.

BACKGROUND: Cross resistance is common among the non-nucleoside reverse transcriptase inhibitors (NNRTIs). G190A appears in 5-15% of the patients treated with nevirapine or efavirenz who develop clinical resistance. OBJECTIVES: In this study we investigated the effect of G190A and other NNRTI substitutions on the phenotypic susceptibility to this class of drugs. STUDY DESIGN: We identified 15 individuals, who after treatment with NNRTIs (nevirapine or efavirenz; median exposure of 20 months), developed isolated G190A, G190A in combination with K103N, or K103N alone. Phenotypic and genotypic analyses of stored plasma specimens were performed before and after the mutations occurred to assess NNRTI susceptibility. RESULTS: All isolates that developed only G190A substitution became less susceptible to nevirapine (median: 125-fold) and efavirenz (median: 10-fold) but were 2.5-fold more sensitive to delavirdine (Wilcoxon P = 0.06). In the group with only K103N substitution, acquisition of resistance to all NNRTIs was observed. In the group with the double substitutions, G190A and K103N, delavirdine susceptibility decreased 13-fold, while resistance to nevirapine and efavirenz decreased by 239- and 154-folds, respectively (Kruskal-Wallis H P = 0.009). CONCLUSIONS: The data suggest that the presence of a G190A substitution attenuates the phenotypic resistance associated with a K103N substitution, although resistance is still present. The in vivo significance of the increased phenotypic susceptibility to delavirdine is not known but could be evaluated in a clinical trial.

Anti-HIV Agents↗
PubMed1992

Mutagenesis of the Glu-89 residue in human immunodeficiency virus type 1 (HIV-1) and HIV-2 reverse transcriptases: effects on nucleoside analog resistance.

A Glu-89-->Gly alteration in the human immunodeficiency virus type 1 reverse transcriptase (RT) was previously shown to result in resistance to several dideoxynucleoside analogs and to phosphonoformic acid (PFA; foscarnet). This residue was altered to Ala, Val, Ser, Thr, Gln, Asp, Asn, or Lys, and the ddGTP and PFA sensitivities of the mutant RTs were measured. Replacements with Ala, Gly, Val, and Thr led to resistance to inhibition by ddGTP, while mutants with amino acid Ser, Gln, Asn, Asp, or Lys displayed only moderate or no resistance. A similar result was obtained with inhibition by PFA, except that the Asp-89 mutant also displayed resistance. Furthermore, the introduction of Glu-89-->Gly alteration into the RT of human immunodeficiency virus type 2 likewise rendered it resistant to both ddGTP and PFA.

Amino Acid Sequence↗
PubMed1989

Point mutations in conserved amino acid residues within the C-terminal domain of HIV-1 reverse transcriptase specifically repress RNase H function.

Two single site substitutions (E478----Q and H539----F) were introduced into the C-terminal RNase H domain of HIV-1 reverse transcriptase. These mutant proteins were expressed in Escherichia coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography. Both enzymes are clearly defective in RNase H function, but exhibit wild type reverse transcriptase activity.

Amino Acid Sequence↗
PubMed1997

Moloney murine leukemia reverse transcriptase suspect in the production of multiple misincorporations during hprt cDNA synthesis.

Our laboratory has characterized several hundred mutant hprt cDNAs produced using Moloney murine leukemia reverse transcriptase to convert mRNA to cDNA. During the characterization of these mutants we have detected six T-lymphocyte mutants that demonstrate multiple G:C --> A:T transitions along the hprt cDNA coding sequence. Attempts to repeat the mRNA to cDNA conversion and subsequent characterization have demonstrated that the multiple transitions are likely artifacts. We suggest that reverse transcriptase is directly responsible for these multiple base substitutions and as such, that multiple mutations be viewed as suspect requiring confirmation at the genomic level.

Animals↗
PubMed2001

Solid-phase synthesis of the alkenyldiarylmethane (ADAM) series of non-nucleoside HIV-1 reverse transcriptase inhibitors.

The Sonogashira and Stille cross-coupling reactions have been employed in the synthesis of several non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the alkenyldiarylmethane (ADAM) series. The synthesis has been carried out both in solution and on a solid support. In contrast to previous syntheses of NNRTIs in the ADAM series, the present strategy allows the incorporation of differently substituted aromatic rings in a stereochemically defined fashion. The most potent of the new ADAMs inhibited the cytopathic effect of HIV-1RF in CEM-SS cell culture with an EC50 value of 20 nM.

Anti-HIV Agents↗
PubMed2004

Hybrids of [TSAO-T]-[foscarnet]: The first conjugate of foscarnet with a non-nucleoside reverse transcriptase inhibitor through a labile covalent ester bond.

This paper describes the first example of combination of non-nucleoside reverse transcriptase inhibitors such as TSAO derivatives and foscarnet (PFA) in a single molecule through a labile covalent ester bond. The essential criteria in the design of these hybrids [TSAO-T]-[PFA] was to explore if the conjugation of foscarnet with the highly lipophilic TSAO derivative may facilitate the penetration of the conjugates through the cell membrane and if the hybrids escape extracellular hydrolysis and regenerate the parent inhibitors intracellulary. Several [TSAO-T]-[PFA] conjugates proved markedly inhibitory to HIV-1. Some of them also showed potent activity against PFA-resistant HIV-1 strains but fewer had detectable inhibitory activity against TSAO-resistant HIV-1 strains. These results indicated a pivotal role of the TSAO component of the hybrid but not the PFA component in the activity of the conjugates. Moreover, stability studies of the [TSAO-T]-[PFA] conjugates demonstrated that the compounds were stable in PBS whereas some of the conjugates regenerated the parent inhibitors in extracts from CEM cells.

Cell Extracts↗
PubMed2002

HEPT derivatives as non-nucleoside inhibitors of HIV-1 reverse transcriptase: QSAR studies agree with the crystal structures.

The interest in the non-nucleoside inhibitors (NNIs) to the reverse transcriptase (RT) as anti-AIDS agents has grown in the last ten years. The compound 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) is the precursor of the most studied class of NNIs, from which hundreds of derivatives have been synthesized and tested. There are at least twelve QSAR studies about the HEPT derivatives as RT inhibitors. Most of the predictions derived by these studies are related to the nature of the active site near the substituents at positions N-1 and C-5, and at the C-6 phenyl ring. The validity of these models has been checked against the 3-D structure of HIV 1 RT-HEPT complexes available. Most of these predictions were confirmed at the molecular level.

Binding Sites↗
PubMed1996

Linked lexitropsins and the in vitro inhibition of HIV-1 reverse transcriptase RNA-directed DNA polymerization: a novel induced-fit of 3,5 m-pyridyl bisdistamycin to enzyme-associated template-primer.

Five classic DNA minor groove-binding drugs and a series of bis-linked lexitropsins based on netropsin and distamycin have been screened for their effectiveness in inhibiting transcription by HIV-1 reverse transcriptase (RT) on a poly(rA).oligo(dT) template-primer (TP). The two most effective drugs, 3,5 m-pyridyl-linked bisdistamycin (MPyr) and trans-vinyl-linked bisdistamycin (TVin), show (1) enhanced inhibition in reactions initiated with pre-incubated enzyme template-primer (ETP) and (2) reduced affinity for a "free" TP analog, when compared with the parent drug distamycin. All three drugs lack the ability to inhibit processive incorporation of nucleotide, suggesting drug intervention instead at initiation or termination of processive cycles. The two bis-linked drugs exhibit different kinetic behavior with reverse transcriptase's two substrates: template-primer and nucleotide. When primer is the variable substrate, TVin is partially noncompetitive and MPyr is dead-end competitive (Ki = 6.5 microM). With nucleotide as substrate, TVin is noncompetitive at low drug concentrations and MPyr is uncompetitive. Gel band mobility shift assays with MPyr indicate that the drug inhibits via entrapment of TP on the enzyme rather than displacement of TP from the enzyme surface. The conformation of nucleic acid is most likely altered upon MPyr binding, enhancing the induced fit of enzyme to hybrid duplex. The relevance of this novel mode of inhibition is considered in relation to enzyme association/dissociation with TP that occurs prior to (-)-DNA strand transfer, and to the structural implications of an enzyme-bound hybrid RNA/DNA nucleic acid.

Antiviral Agents↗
PubMed1992

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Amino Acid Sequence↗
PubMed1998

Structure-based design of novel dihydroalkoxybenzyloxopyrimidine derivatives as potent nonnucleoside inhibitors of the human immunodeficiency virus reverse transcriptase.

Two highly potent dihydroalkoxybenzyloxopyrimidine (DABO) derivatives targeting the nonnucleoside inhibitor (NNI) binding site of human immunodeficiency virus (HIV) reverse transcriptase (RT) have been designed based on the structure of the NNI binding pocket and tested for anti-HIV activity. Our lead DABO derivative, 5-isopropyl-2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-on e, elicited potent inhibitory activity against purified recombinant HIV RT and abrogated HIV replication in peripheral blood mononuclear cells at nanomolar concentrations (50% inhibitory concentration, <1 nM) but showed no detectable cytotoxicity at concentrations as high as 100 microM.

Binding Sites↗
PubMed1995

Site-directed mutagenesis of arginine 72 of HIV-1 reverse transcriptase. Catalytic role and inhibitor sensitivity.

In order to determine the catalytic role of Arg72 of HIV-1 reverse transcriptase (RT), we carried out site-directed mutagenesis at codon 72. Two mutant proteins (R72A and R72K) were purified and characterized. With Arg to Ala substitution the kcat of the polymerase reaction was reduced by nearly 100-fold with poly(rA) template, but only about 5-15-fold with poly(rC) and poly(dC) templates. The Arg to Lys substitution exhibited a qualitatively similar pattern, although the overall reduction in kcat was less severe. Most interestingly, we noted a large difference in the rate constant of the first and second nucleotide incorporation by R72A, suggesting that Arg72 participates in the reaction after the formation of the first phosphodiester bond. We propose this step to be the pyrophosphate binding and removal step following the nucleotidyltransferase reaction. Support for this proposal is obtained from the observation that the R72A mutant (i) exhibited a pronounced translocation defect in the processivity analysis, (ii) lacked the ability to catalyze pyrophosphorolysis, and (iii) showed complete resistance to phosphonoformate, an analog of PPi.Arg72 is the first residue of HIV-1 RT proposed to be involved in the pyrophosphate binding/removal function of RT.

Arginine↗
PubMed1989

Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins.

The expression strategy of the duck hepatitis B virus (DHBV) P gene, which is assumed to encode the viral reverse transcriptase, was investigated by mutational analysis. This study showed that P gene expression starts in the region where the P gene overlaps the viral core gene. However, in contrast to retroviral reverse transcriptases, which are expressed via gag-pol fusion protein intermediates, the DHBV P gene product was found to be synthesized starting at a P gene ATG codon. The resulting protein can complement polymerase-negative mutants in trans and can reverse transcribe viral pregenomic RNA that does not encode an active polymerase. These findings raise the question of how reverse transcription of cellular RNAs can be avoided in infected cells.

Animals↗
PubMed1999

Intramolecular chimeras of the p51 subunit between HIV-1 and FIV reverse transcriptases suggest a stabilizing function for the p66 subunit in the heterodimeric enzyme.

The human immunodeficiency virus (HIV) reverse transcriptase (RT) is a heterodimeric enzyme composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. Recently we showed that p51 plays an important role in the conformation of p66 within the HIV-1 RT heterodimer and hence appears to influence its catalytic activities [Amacker, M., and H ubscher, U. (1998) J. Mol. Biol. 278, 757-765]. This was further investigated here via construction of three intramolecular chimeras of HIV-1 and FIV RTs. The first 25 and 112 amino acids of the N terminus, respectively, as well as the last 22 amino acids of the C terminus in the p51 subunit of HIV-1 RT were exchanged with the corresponding regions of the FIV RT and combined with the wild-type HIV-1 p66. Characterization of these chimeric RT heterodimers demonstrated significant biochemical differences in (i) DNA-dependent DNA synthesis, (ii) strand displacement DNA synthesis, and (iii) RNase H activity. Our results indicate that both the N and C termini of HIV-1 RT p51 appear to be important in stabilizing the RT heterodimer for enzymatic functions.

Amino Acid Sequence↗