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PubMed1996

Synthetic peptides coupled to the lipotripeptide P3CSS induce in vivo B and Thelper cell responses to HIV-1 reverse transcriptase.

To evaluate the ability of the lipotripeptide P3CSS to increase peptide-specific immune responses in vivo, we immunized mice from different inbred strains (BALB/c, C3H/HeJ, C57BL/6) with the 22-mer lipopeptide conjugates P3CSS-[RT-(522-543)] and P3CSS-[RT-(528-549)] of HIV-1 reverse transcriptase (RT) which included an immunodominant Th epitope [i.e. RT-(528-543)] characterized previously. Analysis of T and B cell responses to these lipopeptide conjugates indicated that specific Th responses could be readily induced in vivo. The peptide segments could also efficiently prime mice for secondary recognition of native RT. The use of shorter peptides permitted a delineation of the minimal T cell recognition site of this RT C-terminal region [i.e. RT-(528-540)]. Close to this T cell epitope we identified a B cell determinant containing the motif EQVD [RT-(546-549)] which was recognized in three different strains of mice (H-2b, H-2d and H-2k). A comparison with X-ray analysis of the C-terminal region of HIV-1 reverse transcriptase indicated exposed positions of these Th and B cell epitopes. Both the presence of T and B cell sites and its limited polymorphism make the region RT-(528-549) a promising candidate for vaccine design. The use of the P3CSS adjuvant/carrier principle as a nontoxic adjuvant may be of major importance in the development of vaccines applicable to humans.

Adjuvants, Immunologic↗
PubMed2006

Cytotoxic T cell recognition of an HIV-1 reverse transcriptase variant peptide incorporating the K103N drug resistance mutation.

During HIV-1 infection, cytotoxic T cell (CTL) responses exert strong selective pressure on the replicating virus population. Here we report evidence for T cell activity against the drug resistant K103N region of viral reverse transcriptase in three HIV-1 infected patients exposed to NNRTI antiretroviral drugs. We further characterize the response in one patient by ELISPOT analysis. A nine amino acid peptide incorporating 103N was recognized by patient T cells whereas the wild type was not. The RT K103N mutation is selected by the NNRTI class of HIV drugs. We hypothesize that, in certain individuals, CTL responses against 103N-containing epitopes may protect against NNRTI drug resistance. Characterizing such responses in the context of HLA subtypes could lead to tailored HIV drug therapy or to the design of therapeutic vaccines.

Journal Article↗
PubMed1994

The reverse transcriptase of HIV-1: from enzymology to therapeutic intervention.

The human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Replication of this virus requires the activity of a retrovirus encoded RNA-dependent DNA polymerase, or reverse transcriptase (RT). HIV-1 RT is required for the synthesis of the double-stranded proviral DNA from the single-stranded retroviral RNA genome. HIV-1 RT has two subunits of 66 kDa and 51 kDa. The 66-kDa subunit contains the DNA polymerase and RNase H domains whereas the 51-kDa subunit, obtained by proteolytic maturation of the former subunit, has only the DNA synthetic activity. Two recently reported crystal structures of HIV-1 RT have revealed the very asymmetric structure of this molecule. In addition to providing information concerning the mechanism of nucleic acid polymerization, biochemical and biophysical studies of this enzyme are providing key insights for the design of selective antiviral agents. The multiple activities displayed by reverse transcriptase in the replication of the retroviral genome ensure that this enzyme will remain at the forefront of antiviral strategies in the fight against AIDS and other retrovirus-related pathologies.

HIV Reverse Transcriptase↗
PubMed1991

Analysis of the RNA- and DNA-dependent DNA polymerase activities of point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity.

The RNA- and DNA-dependent DNA polymerase activities of two point mutants of HIV-1 reverse transcriptase lacking ribonuclease H activity have been compared to the wild-type enzyme activities using substrates consisting of an oligodeoxynucleotide primer hybridized to either a RNA or a DNA template. The RNase H phenotype had a negligible effect on the steady-state kinetics and processivity of reverse transcription of a homopolymer template-primer [poly(A).oligo(dT)]. However, analysis of the distribution of DNA products indicated that the ability of the mutants to reverse-transcribe a specifically primed 345-nucleotide heteropolymeric RNA template derived from the gag region of HIV-1 was impaired relative to the wild-type enzyme. Although the wild-type and mutant enzymes shared the same pause sites of synthesis along the RNA template, certain prematurely terminated nascent primer chains were poorly extended by the mutant enzymes and hence accumulated, suggesting that a catalytically functional RNase domain facilitated reinitiation of DNA synthesis at specific pause sites along a heteropolymer template. In contrast, the processivity and product distribution of DNA synthesis directed by a heteropolymer gag DNA template of the same nucleotide sequence were not significantly influenced by the RNase H phenotype of the mutants.

Base Sequence↗
PubMed2004

Interaction of nucleoside inhibitors of HIV-1 reverse transcriptase with the concentrative nucleoside transporter-1 (SLC28A1).

Human concentrative nucleoside transporter-1 (hCNT1) (SLC28A1) is a widely expressed, high-affinity, pyrimidine-preferring, nucleoside transporter implicated in the uptake of naturally occurring pyrimidine nucleosides as well as a variety of derivatives used in anticancer treatment. Its putative role in the uptake of other pyrimidine nucleoside analogues with antiviral properties has not been studied in detail to date. Here, using a hCNT1 stably transfected cell line and the two-electrode voltage-clamp technique, we have assessed the interaction of selected pyrimidine-based antiviral drugs, inhibitors of HIV-1 reverse transcriptase such as zidovudine (AZT), stavudine (d4T), lamivudine (3TC) and zalcitabine (ddC), with hCNT1. hCNT1 transports AZT and d4T with low affinity, whereas 3TC and ddC are not translocated, the latter being able to bind the transporter protein. Selectivity appears to rely mostly upon the presence of a hydroxyl group in the 3'-position of the ribose ring. Thus, hCNT1 cannot be considered a broad-selectivity pyrimidine nucleoside carrier; in fact, very slight changes in substrate structure provoke a dramatic shift in selectivity.

Animals↗
PubMed1993

Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots.

We have observed previously that DNA template-directed polymerization by the type 1 human immunodeficiency virus reverse transcriptase is error-prone for single-nucleotide substitution, addition and deletion errors at homopolymeric sequences. We have also noted strong termination of processive synthesis at these positions (Bebenek, K., Abbotts, J., Roberts, J. D., Wilson, S. H., and Kunkel, T. A. (1989) J. Biol. Chem. 264, 16948-16956). Here we have tested three models to explain errors at these hot spots: template-primer misalignment for deletion errors, and dislocation and direct miscoding for substitution errors. The approach involves introducing single-nucleotide changes within or flanking the homopolymeric hot spots and examining the effects that these changes have on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase error rate, error specificity, and termination probability. The results obtained suggest that single-nucleotide deletion errors in homopolymeric runs result from template-primer misalignment and that both direct miscoding and template-primer dislocation contribute to the base substitution hot spots. The data also suggest that base substitution errors at one position can be templated by the preceding nucleotide or either of the next two nucleotides. Frameshift error rates at homopolymeric sites were affected by changes in the sequences flanking the runs, including single-nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot. Both increases and decreases in frameshift fidelity were observed, and most of these correlated with concomitant increases or decreases in the probability that HIV-1 reverse transcriptase terminated processive synthesis within the run. These data provide further support for a relationship between the frameshift fidelity and the processivity of DNA-dependent DNA synthesis by HIV-1 reverse transcriptase.

Base Sequence↗
PubMed1996

New hydrolyzable tannins, shephagenins A and B, from Shepherdia argentea as HIV-1 reverse transcriptase inhibitors.

Two new hydrolyzable tannins, shephagenins A and B, were isolated along with hippophaenin A and strictinin from the leaf extract of Shepherdia argentea, which showed a remarkable inhibitory activity against human immunodeficiency virus (HIV)-1 reverse transcriptase. Their structures, having a gluconic acid core, have been elucidated on the basis of spectroscopic and chemical methods. The inhibitory effect of the leaf extract on HIV-1 reverse transcriptase was found to be due to tannins, and their activities were stronger than that of (-)-epigallocatechin gallate as a positive control.

Antiviral Agents↗
PubMed1995

Resistance to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives is generated by mutations at multiple sites in the HIV-1 reverse transcriptase.

Virus isolates resistant to 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and a highly potent HEPT derivative, [1-benzyloxymethyl-5-ethyl-6-(alpha-pyridylthio)uracil] (NSC 648400, E-BPTU), were selected in cell culture. Cross-resistance evaluation indicated that the two drug-resistant virus isolates were phenotypically distinct from one another although each of the virus isolates was resistant to both of the HEPT derivatives. The virus isolate resistant to NSC 648400 had a single amino acid change in the reverse transcriptase (Y181C) which resulted in cross-resistance to all of the nonnucleoside reverse transcriptase inhibitors evaluated, with the exception of calanolide A. The NSC 648400-resistant virus isolate exhibited 15-fold enhanced sensitivity to calanolide A. The virus isolate selected in the presence of HEPT exhibited a single amino acid change (P236L) which was not cross-resistant to other nonnucleoside RT inhibitors tested with the exception of the two HEPT derivatives. This HEPT-resistant virus isolate exhibited enhanced sensitivity (5- to 10-fold) to thiazolobenzimidazole. We have used both virus isolates with defined single amino acid changes in the RT and bacterially expressed RTs with site-directed amino acid substitutions to test the effects of a wide variety of mutations on the activity of NSC 648400. Single mutations at amino acids 101, 103, 106, 181, or 236 yielded virus with high resistance (> 20-fold) to NSC 648400, while lower levels of resistance were seen with mutations at amino acids 98, 100, or 108. These results suggest that several changes in the conformation of the nonnucleoside inhibitor binding site of the HIV-1 reverse transcriptase can affect the inhibitory activity of the HEPT class of compounds.

Antiviral Agents↗
PubMed1997

RNase H cleavage of tRNAPro mediated by M-MuLV and HIV-1 reverse transcriptases.

RNA-DNA hybrid model substrates which mimic an intermediate of Moloney murine leukemia virus (M-MuLV) reverse transcription at the stage where the tRNAPro is removed were constructed. This substrate was used to assay the ability of M-MuLV reverse transcriptase (RT) to cleave the RNA portion of the substrate. The cleavage specificities of the cognate M-MuLV RT and the heterologous enzyme from the human immunodeficiency virus type-1 (HIV-1) were compared. M-MuLV and HIV-1 RT recognize and cleave the RNA at distinct positions. The site of the initial RNase H cleavage in vitro was determined using 3' end nearest neighbor analysis of the initial cleavage product. M-MuLV RT/RNase H removed the model tRNAPro between the terminal ribo-A and ribo-C, resulting in a terminal ribo-A attached to the viral DNA, whereas HIV-1 RT/RNase H was shown to cleave at the RNA-DNA junction. Analysis of the DNA over time indicated that the ribo-A is subsequently removed by M-MuLV RT. In vivo analysis from double-LTR circle junctions illustrated that 16 of the 23 clones isolated possessed the predicted junction if complete removal of the tRNA primer were to occur. The predicted junction for complete removal of the tRNA primer was CATT-AATG. One aberrant circle junction was isolated which could result from the use of an alternative primer. In contrast with HIV, no M-MuLV circle junctions were isolated which indicated processing of a single-LTR terminus by integrase. Analysis from in vivo and in vitro studies indicate that the M-MuLV tRNAPro primer is completely removed after plus-strand strong-stop synthesis.

3T3 Cells↗
PubMed2006

HIV-1 reverse transcriptase structure with RNase H inhibitor dihydroxy benzoyl naphthyl hydrazone bound at a novel site.

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 A resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 A away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.

Cell Line, Tumor↗
PubMed1997

Alanine-scanning mutations in the "primer grip" of p66 HIV-1 reverse transcriptase result in selective loss of RNA priming activity.

Alanine-scanning mutants of the primer grip region of human immunodeficiency virus type 1 reverse transcriptase were tested for their ability to extend RNA and DNA versions of the polypurine tract primer, and an oligonucleotide representing the 18-nucleotide sequence at the 3' end of tRNALys3. A majority of the mutant enzymes were either completely or severely deficient in RNA priming activity, but, with only one exception, were able to efficiently extend DNA versions of the same primers. The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activity. Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3' terminus of the polypurine tract, which is required for primer removal. An alanine substitution at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level. Taken together, these results demonstrate for the first time that there are residues in human immunodeficiency virus type 1 reverse transcriptase which are specifically involved in protein-nucleic acid interactions with RNA primers.

Alanine↗
PubMed2001

Quinoxalinylethylpyridylthioureas (QXPTs) as potent non-nucleoside HIV-1 reverse transcriptase (RT) inhibitors. Further SAR studies and identification of a novel orally bioavailable hydrazine-based antiviral agent.

Quinoxalinylethylpyridylthioureas (QXPTs) represent a new class of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 6-FQXPT (6). Docking studies based on the three-dimensional structure of RT prompted the synthesis of novel heteroarylethylpyridylthioureas which were tested as anti-HIV agents. Several compounds proved to be potent broad-spectrum enzyme inhibitors and significantly inhibited HIV-1 replication in vitro. Their potency depends on the substituents and the nature of the heterocyclic skeleton linked to the ethyl spacer, and structure-activity relationships are discussed in terms of the possible interaction with the RT binding site. Although the new QXPTs analogues show potent antiviral activity, none of the compounds tested overcome the pharmacokinetic disadvantages inherent to ethylpyridylthioureidic antiviral agents, which in general have very low oral bioavailability. Through an integrated effort involving synthesis, docking studies, and biological and pharmacokinetic evaluation, we investigated the structural dependence of the poor bioavailability and rapid clearance within the thioureidic series of antivirals. Replacing the ethylthioureidic moiety with a hydrazine linker led to a new antiviral lead, offering promising pharmacological and pharmacokinetic properties in terms of antiviral activity and oral bioavailability.

Animals↗
PubMed1996

Simultaneous determination of L-702,007, a HIV-1 reverse transcriptase inhibitor, and its 6-hydroxy metabolite in human plasma by column-switching high-performance liquid chromatography with fluorescence detection.

A method for the determination of L-702,007, a human immunodeficiency virus type 1 reverse transcriptase inhibitor, and its 6-hydroxy metabolite in human plasma is described. Plasma samples are extracted using phenyl solid-phase extraction columns. The extract is analyzed by HPLC using a column-switching system to remove interferences from late-eluting endogenous components. Fluorescence detection at an excitation wavelength of 314 nm and an emission wavelength of 390 nm is utilized. The assay was validated in the concentration range of 10-200 ng/ml when 1-ml aliquots of plasma were extracted. The assay has been used to support human pharmacokinetic studies.

Biotransformation↗
PubMed2006

TSAO derivatives, inhibitors of HIV-1 reverse transcriptase dimerization: recent progress.

There is an urgent need for the development of new and safer drugs for the treatment of HIV (human immunodeficiency virus) infection, active against the currently resistant viral strains or directed to novel targets in the viral replicative cycle that may be useful for multiple drug combination. TSAO derivatives are a peculiar group of highly functionalized nucleosides that belong to the so-called nonnucleoside RT inhibitors (NNRTIs). HIV-1 reverse transcriptase (RT) is a key enzyme that plays an essential and multifunctional role in the life cycle of the virus and thus represents a key target for antiviral chemotherapeutic intervention. The dimeric form of the enzyme is absolutely required for all enzymatic activities. Thus, the process of dimerization and subsequent maturation into the p66/p51 heterodimer is essential for a fully functional RT and constitutes a target for therapeutic intervention, however to date such agents have not been developed. TSAO molecules are a peculiar group of non-nucleoside RT inhibitors that exert a unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They interact at the p66/p51 heterodimer interface of the enzyme. They were the first small non peptidic molecules shown to interfere with the dimerization process of the enzyme. This review covers the recent work within this family of compounds aimed at enhancing their interaction with the dimer interface of HIV-1 RT.

Binding Sites↗
PubMed2004

Novel benzophenones as non-nucleoside reverse transcriptase inhibitors of HIV-1.

GW4511, GW4751, and GW3011 showed IC50 values < or =2 nM against wild type HIV-1 and <10 nM against 16 mutants. They were particularly potent against NNRTI-resistant viruses containing Y181C-, K103N-, and K103N-based double mutations, which account for a significant proportion of the clinical failure of the three currently marketed NNRTIs. The antiviral data together with the favorable pharmacokinetic data of GW4511 suggested that these benzophenones possess attributes of a new NNRTI drug candidate.

Anti-HIV Agents↗
PubMed1994

HIV-1 reverse transcriptase codon 215 mutation and clinical outcome in children treated with zidovudine.

OBJECTIVE: In HIV-infected adults prolonged monotherapy with zidovudine may be associated with the appearance of HIV strains with decreased zidovudine sensitivity, owing to specific mutations in the reverse transcriptase (RT) gene, and this has been suggested to be a reason for reduced zidovudine efficacy. This study was undertaken to determine the appearance of mutation at codon 215 of the RT gene in proviral DNA from PBMCs in HIV-infected children. DESIGN: A prospective, open study. SETTING: A University Pediatric Department. PATIENTS AND METHODS: Nineteen HIV-infected symptomatic children were treated with zidovudine for a median of 24 months. Clinical and laboratory controls for HIV infection status were performed monthly. Mutant proviral sequences were evaluated at the start of therapy, every 3 months during the first 6 months of therapy, and every 6 months thereafter. Clinical outcome was defined as stable or deteriorating. RESULTS: No child had proviral sequences mutant at codon 215 before starting zidovudine. Ten of 13 children who had received zidovudine for more than 6 months developed mutant proviral sequences. All the children (10 of 10) with mutant proviral sequences had a deteriorating clinical condition, compared to none of those (0 of 9) without mutation at codon 215. CONCLUSION: The appearance of HIV-1 codon 215 mutation seems to be strongly associated with zidovudine therapy and with clinical progression of HIV disease in children.

Base Sequence↗
PubMed1997

Synergistic inhibition of HIV-1 reverse transcriptase by combinations of chain-terminating nucleotides.

Synergistic inhibition of HIV replication in cell culture has been reported for many combinations of reverse transcriptase inhibitors. However, the biochemical basis underlying this interaction is in most cases unknown. It has been previously shown that combinations of L-697,661 or U-90152s with AZT or ddC synergistically inhibit HIV-1 replication in cell culture. The combination of AZT with ddC is also favorable with respect to the inhibition of viral replication. However, the corresponding combinations showed no synergy in inhibiting enzyme activity when tested on conventional polymerase assays using homo- or heteropolymeric RNA and DNA as template. Data obtained suggest that amplification of the effect of chain terminators, a consequence of the high potential number of termination sites present on the template, override the synergistic effect expected for the combination of two independent nucleotide analogs. When a saturating amount of enzyme over template:primer was used, and a single site on the template was available for each chain terminator, the combination of AZTTP and ddCTP synergistically inhibited enzyme activity, whereas, as expected, the combination of AZTTP and ddTTP behaved as merely additive. Under similar conditions the combination of U-90152s and AZTTP was also synergistic. These results suggest that synergy found in antiviral assays with combinations having nucleosidic inhibitors is not related to the synergistic inhibition of reverse transcriptase and might be due to the presence in the viral population of virus strains with different sensitivity to the inhibitors.

Anti-HIV Agents↗
PubMed1996

A photolabile 2',3'-dideoxyuridylate analog bearing an aryl(trifluoromethyl)diazirine moiety: photoaffinity labeling of HIV-1 reverse transcriptase.

In order to develop a photoaffinity labeling reagent for DNA polymerases, including retroviral reverse transcriptase (RT), we utilized 2',3'-dideoxy-E-5-[4-(3-trifluoromethyl-3H-diazirin-3-yl) styryl]UTP (TDSddUTP) as a substrate dTTP analog. Photoaffinity labeling experiments with human immunodeficiency virus type-1 (HIV-1) RT using a radioactive labeling reagent ([gamma-32P]TDSddUTP) and poly(A).oligo(dT) as the template/primer yielded different results depending on the concentration of Mg2+. In the presence of 0.025 mM Mg2+, photoaffinity labeling showed that TDSddUTP bound selectively to the dTTP binding site in the 66 kDa subunit of the p66/p51 heterodimeric enzyme protein when irradiated by near-UV light (365 nm). In the presence of 4 mM Mg2+ or 0.05 mM Mn2+, TDSddUTP was incorporated into the 3'-end of the primer strand due to RT activity and the resulting photolabile primer bound to the 66 kDa subunit of HIV-1 RT on photoirradiation. These results suggest that TDSddUTP could be a useful tool for studying the substrate binding site(s) of DNA polymerases, including HIV-1 RT, which show affinity for this compound.

Affinity Labels↗