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PubMed2003

Negative effect of the M184V mutation in HIV-1 reverse transcriptase on initiation of viral DNA synthesis.

The M184V mutation in HIV reverse transcriptase (RT) is associated with high-level resistance against the nucleoside inhibitor lamivudine as well as diminished viral replication capacity. We have previously demonstrated that HIV variants containing the M184V mutation were relatively unable to successfully undergo compensatory mutagenesis following deletion of an A-rich loop located upstream of the primer binding site (PBS). To understand the mechanisms involved, we synthesized viral RNA templates containing different compensatory mutations that were emergent during the long-term culture of the A-rich loop-deleted viruses. These templates were then used in cell-free reverse transcription initiation assays and in tRNA primer placement assays performed with either recombinant wild-type RT or recombinant RT containing the M184V substitution. The results showed that the RNA template that contained the A-rich loop deletion was impaired in ability to initiate reverse transcription and that the presence of the M184V substitution in RT amplified this effect. Clearance from pausing at position +3 during synthesis of viral DNA was identified as a sensitive step in this reaction that could not be efficiently bypassed with the M184V mutant enzyme. Increased efficiency of initiation was seen with the deleted RNA templates that also contained mutations identified in the revertant viruses, provided that these mutations facilitated formation of a competent binary tRNA/RNA complex. These findings provide biochemical evidence that initiation of tRNA(Lys3)-primed DNA synthesis is an important rate-limiting step in reverse transcription that correlates with viral replication fitness.

DNA Primers↗

PubMed2005

Dissecting the effects of DNA polymerase and ribonuclease H inhibitor combinations on HIV-1 reverse-transcriptase activities.

Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.

Anti-HIV Agents↗

PubMed1995

The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design.

BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

Benzodiazepines↗

PubMed2006

Synthesis and anti-HIV activity evaluation of 1-[(alkenyl or alkynyl or alkyloxy)methyl]-5-alkyl-6-(1-naphthoyl)-2,4-pyrimidinediones as novel non-nucleoside HIV-1 reverse transcriptase inhibitors.

The synthesis and anti-HIV activity evaluation of a new series of 2,4-pyrimidinediones bearing a 6-(1-naphthoyl) group are described. In general, it was found that most of the title compounds showed good activities against human immunodeficiency virus type-1 (HIV-1). In particular, compound 26 displayed the most potent anti-HIV-1 activity (IC(50)=0.11+/-0.05 microM), inhibiting HIV-1 replication in MT-4 cells more effectively than HEPT (by 45-fold) and DDI (by 50-fold).

Alkenes↗

PubMed2000

RNA motifs mediating in vivo site-specific nonhomologous recombination in (+) RNA virus enforce in vitro nonhomologous crossovers with HIV-1 reverse transcriptase.

There are several lines of evidence that both RNA viruses and retroviruses recombine according to a copy choice mechanism. Using the brome mosaic virus (BMV)-based system, we recognized elements in the RNA structure that enhance nonhomologous crossovers within or near the local heteroduplex formed by recombining molecules. The same structural motifs were employed in vitro to test the ability of human immunodeficiency virus reverse transcriptase (HIV-RT) to switch templates during DNA synthesis. We demonstrated that a specific combination of the local double-stranded region with short homologous sequences and a hairpin structure allows template switching by HIV-RT. In contrast to BMV replicase, HIV-RT does not mediate the detectable level of recombination using only the heteroduplex structure, though local hybridization between RNA molecules efficiently pauses primer extension. Moreover, the presented data suggest that a proper arrangement of identified structural motifs can ensure site specificity of RNA-RNA recombination. These results indicate that HIV-RT utilizes the same or a very similar mechanism as BMV replicase to change nonhomologous RNA templates in a site-specific manner.

Base Sequence↗

PubMed1994

Fidelity of HIV-1 reverse transcriptase copying a hypervariable region of the HIV-1 env gene.

The unusually high mutation frequency exhibited by the human immunodeficiency virus (HIV) is a major impediment to developing effective vaccines against the virus and to designing analogs that inhibit viral replication. To investigate the molecular basis of HIV hypermutability, we established cell-free assays to measure the fidelity of HIV-1 reverse transcriptase (RT) in copying either DNA or both RNA and DNA templates that contain the hypervariable region 1 of the HIV-1 env gene (V-1). The fidelity of DNA synthesis was measured by repetitively copying the envelope gene (V-1) DNA by HIV-1 RT, followed by cloning and sequencing these newly synthesized DNA products. We found that the error rate of HIV RT copying either RNA or DNA of the env V-1 region is about one misincorporation per 5 kb polymerized. This rate is similar to that found with the M13mp2 forward mutation assay using the lacZ alpha gene as a template. This similarity suggests that the HIV env hypervariable sequence is not inherently hypermutable. The high error rate of HIV RT suggests that misincorporation by this enzyme is a major source of mutations throughout the viral genome and a determinant for rapid viral evolution. The spectrum of mutations produced by HIV RT in vitro partially correlates with the spectrum of HIV mutations observed in AIDS patients. The differences between these spectra highlight the contribution of phenotypic selection during HIV-1 infection. The overall uniformity of misincorporation of HIV-1 RT further suggests an alternative anti-HIV strategy based on increasing viral mutagenesis by nucleotide analogs.

Base Sequence↗

PubMed1994

Premature strand transfer by the HIV-1 reverse transcriptase during strong-stop DNA synthesis.

Reverse transcription of retroviral genomes starts near the 5' end of the viral RNA by use of an associated tRNA primer. According to the current model of reverse transcription, the initial cDNA product, termed minus-strand strong-stop DNA, 'jumps' to a repeated sequence (R region) at the 3' end of the RNA template. The human retroviruses have relatively long R regions (97-247 nucleotides) when compared to murine and avian viruses (16-68 nucleotides). This suggests that the full complement of the R region is not required for strand transfer and that partial cDNA copies of the 5' R can prematurely jump to the 3' R. To test this hypothesis, we generated mutants of the human immunodeficiency virus with R region changes and analyzed whether 5' or 3' R sequences were inherited by the progeny. We found that in most cases, 5' R-encoded sequences are dominant, which is consistent with the model of reverse transcription. Using a selection protocol, however, we were also able to identify progeny viruses with R sequences derived from the original 3' R element. These results suggest that partial strong stop cDNAs can be transferred with R region homologies much shorter than 97 nucleotides.

Base Sequence↗

PubMed2004

Artificial neural networks: non-linear QSAR studies of HEPT derivatives as HIV-1 reverse transcriptase inhibitors.

Structure-anti HIV activity relationships were established for a sample of 80 1-[2-hydroxyethoxy-methyl]-6-(phenylthio)thymine (HEPT) using a three-layer neural network (NN). Eight structural descriptors and physicochemical variables were used to characterize the HEPT derivatives under study. The network's architecture and parameters were optimized in order to obtain good results. All the NN architectures were able to establish a satisfactory relationship between the molecular descriptors and the anti-HIV activity. NN proved to give better results than other models in the literature. NN have been shown to be particularly successful in their ability to identify non-linear relationships.

Anti-HIV Agents↗

PubMed2000

Comparison of a point mutation assay with a line probe assay for the detection of the major mutations in the HIV-1 reverse transcriptase gene associated with reduced susceptibility to nucleoside analogues.

This study compares the performance of a line probe assay (LiPA) for the detection of the major mutations associated with reduced sensitivity to nucleoside analogues with a well characterised point mutation assay (PMA). Plasma samples obtained from patients in a trial of four reverse transcriptase inhibitors (MRC Quattro Trial) were tested by both LiPA and PMA at baseline, 32nd and 64th weeks for the presence of drug resistance associated mutations in the reverse transcriptase (RT) gene. HIV-1 RNA was extracted from plasma by the Boom method and amplified by RT-PCR prior to being tested by LiPA or PMA. Assay discrepancies were further investigated by sequencing of the RT gene. Of 275 samples available from 98 trial subjects, 246 samples were successfully amplified by PCR and analysed by LiPA and PMA for six mutations. Of the 1476 individual codons analysed, LiPA successfully assayed 1444 (97.8%) and PMA gave a result with 1418 (96.1%). LiPA failed to give a result for 32 codons from 22 samples and PMA failed with 58 codons from 38 samples. Gross differences between the two assays, in which one scored a codon as wild-type only and the other as mutant only or vice versa, occurred at 28 codons analysed (1.9%) representing 26 samples from 20 subjects. Sequencing of 22 of the 26 samples confirmed the LiPA result in nine cases, the PMA result in 11 and detected a novel variant at codon 215 in four cases. The PMA and LiPA approach to the detection of the major mutations that are genotypically associated with reduced sensitivity to nucleoside analogues can correctly detect mutations in 97% of the cases.

Acetamides↗

PubMed2005

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones.

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.7 microM, respectively. In contrast, the related tropolone analog beta-thujaplicin (2-hydroxy-4-(methylethyl)-2,4,6-cycloheptatrien-1-one), which lacks the 7-OH group of the heptatriene ring, was inactive, while manicol, which possesses a 7-OH group, inhibited HIV-1 and E.coli RNases H with IC50 = 1.5 microM and 40 microM, respectively. Such a result highlights the importance of the 2,7-dihydroxy function of these tropolone analogs, possibly through a role in metal chelation at the RNase H active site. Inhibition of HIV-2 RT-associated RNase H indirectly indicates that these compounds do not occupy the nonnucleoside inhibitor-binding pocket in the vicinity of the DNA polymerase domain. Both beta-thujaplicinol and manicol failed to inhibit DNA-dependent DNA polymerase activity of HIV-1 RT at a concentration of 50 microM, suggesting that they are specific for the C-terminal RNase H domain, while surface plasmon resonance studies indicated that the inhibition was not due to intercalation of the analog into the nucleic acid substrate. Finally, we have demonstrated synergy between beta-thujaplicinol and calanolide A, a nonnucleoside inhibitor of HIV-1 RT, raising the possibility that both enzymatic activities of HIV-1 RT can be simultaneously targeted.

Anti-HIV Agents↗

PubMed1993

Kinetics of deoxyribonucleotide insertion and extension at abasic template lesions in different sequence contexts using HIV-1 reverse transcriptase.

Deoxyribonucleotide insertion efficiencies were measured opposite site-directed abasic template lesions using human immunodeficiency virus 1 reverse transcriptase (HIV-1RT), and the efficiencies to continue primer synthesis beyond the lesion, by addition of the "next correct" deoxynucleotide, were measured as a function of sequence context. Insertion of purines was favored over pyrimidines, A > G > T approximately C. Primer extension past the lesion occurred by two distinct mechanisms, either by direct or by misalignment extension. An "A-rule" appeared to hold for the case of direct extension, where the abasic template moiety is intrahelical, aligned opposite the primer 3'-terminus. In misalignment extension, the primer terminus is realigned from a site directly opposite the lesion to a new position opposite a neighboring template base 5' to the lesion. Direct extension efficiencies were measured in 16 different configurations, by varying 4 bases at the primer 3'-termini and 4 at the 5'-side (downstream) of the lesion. The predominant order of direct extension was A > G > T approximately C, similar to that observed for insertion. Reduced primer extension rates were not caused by a reduction in HIV-1 RT-DNA binding. Primers terminating in C showed inefficient direct extension, but were readily extended via misaligned configurations. The ratios of direct-to-misalignment extension efficiencies were 27:1, 2.5:1, and 1:25 for A, G, and C opposite the lesion, respectively. For the case of primers terminating in T, misalignment extension was not observed. A striking result was that while primers were extended past an abasic lesion by HIV-1 RT in both direct and misalignment modes, avian myeloblastosis virus RT failed to catalyze significant extension by either mode.

Animals↗

PubMed1995

Novel non-nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. 4. 2-Substituted dipyridodiazepinones as potent inhibitors of both wild-type and cysteine-181 HIV-1 reverse transcriptase enzymes.

The major cause of viral resistance to the potent human immunodeficiency virus type 1 reverse transcriptase (RT) inhibitor nevirapine is the mutation substituting cysteine for tyrosine-181 in RT (Y181C RT). An evaluation, against Y181C RT, of previously described analogs of nevirapine revealed that the 2-chlorodipyridodiazepinone 16 is an effective inhibitor of this mutant enzyme. The detailed examination of the structure-activity relationship of 2-substituted dipyridodiazepinones presented below shows that combined activity against the wild-type and Y181C enzymes is achieved with aryl substituents at the 2-position of the tricyclic ring system. In addition, the substitution pattern at C-4, N-5, and N-11 of the dipyridodiazepinone ring system optimum for inhibition of both wild-type and Y181C RT is no longer the 4-methyl-11-cyclopropyl substitution preferred against the wild-type enzyme but rather the 5-methyl-11-ethyl (or 11-cyclopropyl) pattern. The more potent 2-substituted dipyridodiazepinones were evaluated against mutant RT enzymes (L100I RT, K103N RT, P236L RT, and E138K RT) that confer resistance to other non-nucleoside RT inhibitors, and compounds 42, 62, and 67, with pyrrolyl, aminophenyl, and aminopyridyl substituents, respectively, at the 2-position, were found to be effective inhibitors of these mutant enzymes also.

Cell Line↗

PubMed1998

Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV-1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide.

The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence. A stable ternary elongation complex was assembled containing enzyme, DNA template (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodideoxyuridine triphosphate was incorporated uniquely at the 3' terminus of the 32P-labeled DNA product. Upon photolysis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either trypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separated, purified, and finally subjected to Edman microsequencing. A unique radioactive hexapeptide (V276RQLCK281) was identified and sequenced. Our photoaffinity labeling results were positioned on the HIV-1 RT. DNA.Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site.

Binding Sites↗

PubMed2001

Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP.

The interaction of dNTPs with the active site of HIV-1 reverse transcriptase (HIV RT) has been investigated. The kinetic parameters of primer elongation catalyzed by wild-type HIV-1 RT and two of its mutants with substitutions for Tyr115 using dTTP and two of its photoreactive analogs were determined. The substitution for Tyr115 with alanine or tryptophan resulted in an increase in K(m) values of dTTP and its analogs. Wild-type RT and its mutants were photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photocross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensitizer. The use of the sensitizer decreased the number of modification products and increased selective labeling of the catalytic subunit of both the mutant and wild-type RT.

Alanine↗

PubMed1999

Pharmacokinetics of [(14)C]abacavir, a human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor, administered in a single oral dose to HIV-1-infected adults: a mass balance study.

Abacavir (1592U89) ((-)-(1S, 4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene- 1-m ethanol) is a 2'-deoxyguanosine analogue with potent activity against human immunodeficiency virus (HIV) type 1. To determine the metabolic profile, routes of elimination, and total recovery of abacavir and metabolites in humans, we undertook a phase I mass balance study in which six HIV-infected male volunteers ingested a single 600-mg oral dose of abacavir including 100 microCi of [(14)C]abacavir. The metabolic disposition of the drug was determined through analyses of whole-blood, plasma, urine, and stool samples, collected for a period of up to 10 days postdosing, and of cerebrospinal fluid (CSF), collected up to 6 h postdosing. The radioactivity from abacavir and its two major metabolites, a 5'-carboxylate (2269W93) and a 5'-glucuronide (361W94), accounted for the majority (92%) of radioactivity detected in plasma. Virtually all of the administered dose of radioactivity (99%) was recovered, with 83% eliminated in urine and 16% eliminated in feces. Of the 83% radioactivity dose eliminated in the urine, 36% was identified as 361W94, 30% was identified as 2269W93, and 1.2% was identified as abacavir; the remaining 15.8% was attributed to numerous trace metabolites, of which <1% of the administered radioactivity was 1144U88, a minor metabolite. The peak concentration of abacavir in CSF ranged from 0.6 to 1.4 microg/ml, which is 8 to 20 times the mean 50% inhibitory concentration for HIV clinical isolates in vitro (0.07 microg/ml). In conclusion, the main route of elimination for oral abacavir in humans is metabolism, with <2% of a dose recovered in urine as unchanged drug. The main route of metabolite excretion is renal, with 83% of a dose recovered in urine. Two major metabolites, the 5'-carboxylate and the 5'-glucuronide, were identified in urine and, combined, accounted for 66% of the dose. Abacavir showed significant penetration into CSF.

Adult↗

PubMed2005

Aptamers directed to HIV-1 reverse transcriptase display greater efficacy over small hairpin RNAs targeted to viral RNA in blocking HIV-1 replication.

RNA molecules can be powerful inhibitors of HIV-1 replication. To determine the relative efficacy of siRNAs and RNA aptamers, a direct comparison of three anti-HIV reverse transcriptase aptamers and three shRNAs targeted to HIV-1(R3b) was made. U6 promoter-driven anti-HIV genes were delivered into CEMx174 cells via a retroviral vector, and transduced cells were sorted out via green fluorescent protein function and challenged with HIV. The results show that, at low virus input, shRNAs can block HIV as efficiently as aptamers. When expressed in target cells, both classes of inhibitors blocked early events of reverse transcription, suggesting they are both able to access intracellular reverse transcription complexes. However, at higher multiplicities of infection (m.o.i. of 50), while the aptamers could efficiently inhibit HIV replication, shRNAs did not. RNase protection assays indicated similar steady-state levels or nucleocytoplasmic distribution showing that the differential efficacy was not a reflection of intracellular concentration. The higher potency of anti-RT aptamers could be due to their ability to inhibit two successive rounds of reverse transcription owing to their unique ability to be encapsidated into virion particles. Furthermore, anti-RT aptamers expressed in T cells afforded protection against high-dose infection by chimeric RT-SHIV viruses.

Base Pairing↗

PubMed1990

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys).

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.

Base Sequence↗

PubMed1992

The observed inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate for HIV-1 reverse transcriptase depends on the length of the poly(rA) region of the template.

The inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) against HIV-1 reverse transcriptase (HIV-1 RT) has been further evaluated. The results indicate that the previously reported low Ki values for AZTTP against HIV-1 RT (2.35 nM) are due neither to the to the direct tight binding of AZTTP to HIV-1 RT nor to the interaction of the enzyme with AZTMP moiety terminated primer-templates, but instead they are an artifact of the use of a homotemplate-primer [poly(rA).oligo(dT)]. With a set of RNAs of defined sequence as templates, we demonstrate that the observed Ki value for AZTTP depends on the length of the poly(rA) region following the primer in the RNA template. The more adenosyl residues in the RNA template that are available for processive incorporation of TMP moieties, the lower is the observed Ki value for AZTTP. Since the potencies of new inhibitors of HIV-1 RT are usually compared with that for AZTTP, these results have important consequences for the process of discovery of new HIV inhibitors that are of potential use in AIDS therapy.

Base Sequence↗