Quinolinonyl Non-Diketo Acid Derivatives as Inhibitors of HIV-1 Ribonuclease H and Polymerase Functions of Reverse Transcriptase.

Novel anti-HIV agents are still needed to overcome resistance issues, in particular inhibitors acting against novel viral targets. The ribonuclease H (RNase H) function of the reverse transcriptase (RT) represents a validated and promising target, and no inhibitor has reached the clinical pipeline yet. Here, we present rationally designed non-diketo acid selective RNase H inhibitors (RHIs) based on the quinolinone scaffold starting from former dual integrase (IN)/RNase H quinolinonyl diketo acids. Several derivatives were synthesized and tested against RNase H and viral replication and found active at micromolar concentrations. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, and Mg2+ titration experiments demonstrated that our compounds coordinate the Mg2+ cofactor and interact with amino acids of the RNase H domain that are highly conserved among naïve and treatment-experienced patients. In general, the new inhibitors influenced also the polymerase activity of RT but were selective against RNase H vs the IN enzyme.

Determinants of Active-Site Inhibitor Interaction with HIV-1 RNase H.

The ribonuclease H (RNH) activity of HIV-1 reverse transcriptase (RT) is essential for viral replication and can be a target for drug development. Yet, no RNH inhibitor to date has substantial antiviral activity to allow advancement into clinical development. Herein, we describe our characterization of the detailed binding mechanisms of RNH active-site inhibitors, YLC2-155 and ZW566, that bind to the RNH domain through divalent metal ions, using NMR, molecular docking, and quantum mechanical calculations. In the presence of Mg2+, NMR spectra of RNH exhibited split (two) resonances for some residues upon inhibitor binding, suggesting two binding modes, an observation consistent with the docking results. The relative populations of the two binding conformers were independent of inhibitor or Mg2+ concentration, with one conformation consistently more favored. In our docking study, one distinctive pose of ZW566 showed more interactions with surrounding residues of RNH compared to the analogous binding pose of YLC2-155. Inhibitor titration experiments revealed a lower dissociation constant for ZW566 compared to YLC2-155, in agreement with its higher inhibitory activity. Mg2+ titration data also indicated a stronger dependence on Mg2+ for the RNH interaction with ZW566 compared to YLC2-155. Combined docking and quantum mechanical calculation results suggest that stronger metal coordination as well as more protein-inhibitor interactions may account for the higher binding affinity of ZW566. These findings support the idea that strategies for the development of potent competitive active site RNH inhibitors should take into account not only metal-inhibitor coordination but also protein-inhibitor interaction and conformational selectivity.

Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H.

The rapid emergence of drug-resistant variants is one of the most common causes of highly active antiretroviral therapeutic (HAART) failure in patients infected with HIV-1. Compared with the existing HAART, the recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) is an efficient approach to counteract the failure of anti-HIV treatment due to drug resistance. However, the binding mode and potential resistance profile of these inhibitors with important mechanistic principles remain poorly understood. To address this issue, an integrated computational method was employed to investigate the binding mode of inhibitor JMC6F with HIV-1 IN and RNase H. By using per-residue binding free energy decomposition analysis, the following residues: Asp64, Thr66, Leu68, Asp116, Tyr143, Gln148 and Glu152 in IN, Asp443, Glu478, Trp536, Lys541 and Asp549 in RNase H were identified as key residues for JMC6F binding. And then computational alanine scanning was carried to further verify the key residues. Moreover, the resistance profile of the currently known major mutations in HIV-1 IN and 2 mutations in RNase H against JMC6F was predicted by in silico mutagenesis studies. The results demonstrated that only three mutations in HIV-1 IN (Y143C, Q148R and N155H) and two mutations in HIV-1 RNase H (Y501R and Y501W) resulted in a reduction of JMC6F potency, thus indicating their potential role in providing resistance to JMC6F. These data provided important insights into the binding mode and resistance profile of the inhibitors with a pyrrolyl diketo acid scaffold in HIV-1 IN and RNase H, which would be helpful for the development of more effective dual HIV-1 IN and RNase H inhibitors.

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

The ß1'-ß2' Motif of the RNase H Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Is Responsible for Conferring Open Conformation to the p66 Subunit by Displacing the Connection Domain from the Polymerase Cleft.

The heterodimeric human immunodeficiency virus type 1 reverse transcriptase is composed of p66 and p51 subunits. While in the p51 subunit, the connection domain is tucked in the polymerase cleft; it is effectively displaced from the cleft of the catalytically active p66 subunit. How is the connection domain relocated from the polymerase cleft of p66? Does the RNase H domain have any role in this process? To answer this question, we extended the C-terminal region of p51 by stepwise addition of N-terminal motifs of RNase H domain to generate p54, p57, p60, and p63 derivatives. We found all of the C-terminal extended derivatives of p51 assume open conformation, bind to the template-primer, and catalyze the polymerase reaction. Glycerol gradient ultracentrifugation analysis showed that only p54 sedimented as a monomer, while other derivatives were in a homodimeric conformation. We proposed a model to explain the monomeric conformation of catalytically active p54 derivative carrying additional 21-residues long ß1'-ß2' motif from the RNase H domain. Our results indicate that the ß1'-ß2' motif of the RNase H domain may be responsible for displacing the connection domain from the polymerase cleft of putative monomeric p66. The unstable elongated p66 molecule may then readily dimerize with p51 to assume a stable dimeric conformation.

Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors.

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.

Variability and Signatures of RNase-H Amino Acid Domain and Central Polypurine Tract of HIV-1 B-Subtype from Drug-Naive Individuals.

BACKGROUND: The conversion to HIV-1 single-stranded RNA into double-stranded DNA for nuclear integration is an essential viral step in replication: this process is mediated by Reverse-Transcriptase (RT) and by central polypurine tract (cPPT), a domain where the plus-strand synthesis requires viral primers produced by RNase-H cleavage. Recent studies highlighted the need of investigating the role of RNase-H in RT nucleoside-inhibitors-resistance, because specific mutation(s) could affect cPPT removal and RNase-H cleavage specificity. Thus, the variability of RNase-H and cPPT were studied. METHODS: HIV-1 subtype-B sequences from 746 drug-naïve and 806 antiretroviral-(ARV)-treated patients were used and analysed. RESULTS: In drug-naïve patients, among 54 RNase-H variable residues, 25 were mutated in >5% of patients, and 7 of them were highly variable (>25%), whilst in ARV-treated individuals, 53 RNase-H variable residues were observed, which 24 were mutated in >5% of patients and 6 of them were highly variable (>25%). Differently, a high conservation was observed in cPPT-area, with no statistically significant differences observed between the two datasets analysed. Nevertheless, in ARV-treated patients the variability of cPPT nucleotide at position 6 was found three times higher with respect to the drug-naïve dataset. The topology of the dendrogram has revealed the existence of a cluster (boostrap=0.98) grouping the A6GcPPT with V531I and S519N RNase-H signatures. CONCLUSION: These signatures observed within cPPT and mostly in RNase-H, warrant advanced structural analysis to delineate their potential roles in the affinity/recognition of RT and the cleavage capacity of RNase-H. Exploring further the implications such changes may have on drug-resistance may be relevant.

Major groove binding track residues of the connection subdomain of human immunodeficiency virus type 1 reverse transcriptase enhance cDNA synthesis at high temperatures.

At high temperatures, RNA denaturation can improve the efficiency and specificity of reverse transcription. Refined structures and molecular models of HIV-1 reverse transcriptases (RTs) from phylogenetically distant clades (i.e., group M subtype B and group O) revealed a major interaction between the template-primer and the Arg³58-Gly³59-Ala³6° triad in the large subunit of HIV-1M/B RT. However, fewer contacts were predicted for the equivalent Lys³58-Ala³59-Ser³6° triad of HIV-1O RT and the nucleic acid. An engineered HIV-1O K358R/A359G/S360A RT showed increased cDNA synthesis efficiency above 68 °C, as determined by qualitative and quantitative reverse transcription polymerase chain reactions. In comparison with wild-type HIV-1O RT, the mutant enzyme showed higher thermal stability but retained wild-type RNase H activity. Mutations that increased the accuracy of HIV-1M/B RTs were tested in combination with the K358R/A359G/S360A triple mutation. Some of them (e.g., F61A, K65R, K65R/V75I, and V148I) had a negative effect on reverse transcription efficiency above 65 °C. RTs with improved DNA binding affinities also showed higher cDNA synthesis efficiencies at elevated temperatures. Two of the most thermostable RTs (i.e., mutants T69SSG/K358R/A359G/S360A and K358R/A359G/S360A/E478Q) showed moderately increased fidelity in forward mutation assays. Our results demonstrate that the triad of Arg³58, Gly³59, and Ala³6° in the major groove binding track of HIV-1 RT is a major target for RT stabilization, and most relevant for improving reverse transcription efficiency at high temperatures.

Exploiting drug-resistant enzymes as tools to identify thienopyrimidinone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H.

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3',4'-dihydroxyphenyl (catechol) substituted thienopyrimidinones with submicromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5 °C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthen the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy.

Altered error specificity of RNase H-deficient HIV-1 reverse transcriptases during DNA-dependent DNA synthesis.

Asp(443) and Glu(478) are essential active site residues in the RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). We have investigated the effects of substituting Asn for Asp(443) or Gln for Glu(478) on the fidelity of DNA-dependent DNA synthesis of phylogenetically diverse HIV-1 RTs. In M13mp2 lacZα-based forward mutation assays, HIV-1 group M (BH10) and group O RTs bearing substitutions D443N, E478Q, V75I/D443N or V75I/E478Q showed 2.0- to 6.6-fold increased accuracy in comparison with the corresponding wild-type enzymes. This was a consequence of their lower base substitution error rates. One-nucleotide deletions and insertions represented between 30 and 68% of all errors identified in the mutational spectra of RNase H-deficient HIV-1 group O RTs. In comparison with the wild-type RT, these enzymes showed higher frameshift error rates and higher dissociation rate constants (koff) for DNA/DNA template-primers. The effects on frameshift fidelity were similar to those reported for mutation E89G and suggest that in HIV-1 group O RT, RNase H inactivation could affect template/primer slippage. Our results support a role for the RNase H domain during plus-strand DNA polymerization and suggest that mutations affecting RNase H function could also contribute to retrovirus variability during the later steps of reverse transcription.

A short hairpin loop-structured oligodeoxynucleotide targeting the virion-associated RNase H of HIV inhibits HIV production in cell culture and in huPBL-SCID mice.

BACKGROUND: We have recently demonstrated that an oligodeoxynucleotide (ODN) can enter HIV particles and form a local hybrid at the highly conserved polypurine tract (PPT), the target site of the ODN. This hybrid is recognized by the retrovirus-specific RNase H, which is a virion-associated enzyme. It cleaves the RNA at local hybrids and thereby destroys viral infectivity. This mechanism has been described previously in a mouse model using an oncogenic retrovirus and was commented as driving HIV into suicide. The RNase H is one of four retrovirus-specific enzymes and not yet targeted by antiviral drugs. AIMS: We wanted to analyze the tendency of ODNs to induce mutations in cell culture and its efficacy to inhibit HIV in humanized SCID mice. METHOD: We used cultures of CD4+ T cells infected with HIV-1 after serial passage in the presence of ODNs in the supernatant for up to 3 months, using Foscarnet as positive control, and treated HIV-infected huPBL-SCID mice repeatedly with ODN. RESULTS: Treatment with ODN did not induce mutations of the PPT or the reverse transcriptase polymerase domain in vitro, whereas Foscarnet did. We furthermore demonstrate that ODNs inhibit HIV-1 replication in humanized HIV-infected SCID mice.

Mutagenesis of human immunodeficiency virus reverse transcriptase p51 subunit defines residues contributing to vinylogous urea inhibition of ribonuclease H activity.

The vinylogous urea, NSC727447, was proposed to allosterically inhibit ribonuclease H (RNase H) activity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of its non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied that inhibitor binding altered active site geometry, whereas protein footprinting suggested a contribution from α-helix I residues Cys-280 and Lys-281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys-275 and Thr-286 (comprising α-helix I and portions of the neighboring αH/αI and αI/αJ connecting loops) was combined with a limited vertical scan of Cys-280. A contribution from Cys-280 was strengthened by our observation that all substitutions at this position rendered selectively mutated, reconstituted p66/p51 heterodimers ∼45-fold less sensitive to inhibition. An ∼19-fold reduced IC(50) for p51 mutant T286A coupled with a 2-8-fold increased IC(50) when intervening residues were substituted supports our original proposal of p51 α-helix I as the vinylogous urea binding site. In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product α-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. Finally, reduced DNA strand-transfer activity together with increased vinylogous urea sensitivity of p66/p51 heterodimers containing short p51 C-terminal deletions suggests an additional role for the p51 C terminus in nucleic acid binding that is compromised by inhibitor binding.

Effects of new quinizarin derivatives on both HCV NS5B RNA polymerase and HIV-1 reverse transcriptase associated ribonuclease H activities.

Human immunodeficiency virus 1 (HIV-1) and Hepatitis C virus (HCV) affect 60 and 170 million infected individuals worldwide, respectively, and co-infection by both pathogens is often observed. This represents a serious public health problem that requires the identification of new drugs targeting essential phases of the life cycle of these two viruses. In this report, the synthesis and inhibitory activity of quinizarin derivatives towards both HCV NS5B polymerase and HIV-1 reverse transcriptase associated functions are reported. Our results demonstrate that anthraquinone derivatives are promising anti-polymerase viral inhibitors.

Synthesis, activity, and structural analysis of novel α-hydroxytropolone inhibitors of human immunodeficiency virus reverse transcriptase-associated ribonuclease H.

The α-hydroxytroplone, manicol (5,7-dihydroxy-2-isopropenyl-9-methyl-1,2,3,4-tetrahydro-benzocyclohepten-6-one), potently and specifically inhibits ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV RT) in vitro. However, manicol was ineffective in reducing virus replication in culture. Ongoing efforts to improve the potency and specificity over the lead compound led us to synthesize 14 manicol derivatives that retain the divalent metal-chelating α-hydroxytropolone pharmacophore. These efforts were augmented by a high resolution structure of p66/p51 HIV-1 RT containing the nonnucleoside reverse transcriptase inhibitor (NNRTI), TMC278 and manicol in the DNA polymerase and RNase H active sites, respectively. We demonstrate here that several modified α-hydroxytropolones exhibit antiviral activity at noncytotoxic concentrations. Inclusion of RNase H active site mutants indicated that manicol analogues can occupy an additional site in or around the DNA polymerase catalytic center. Collectively, our studies will promote future structure-based design of improved α-hydroxytropolones to complement the NRTI and NNRTI currently in clinical use.

A combined genotypic and phenotypic human immunodeficiency virus type 1 recombinant virus assay for the reverse transcriptase and integrase genes.

With the approval of the first HIV-1 integrase inhibitor raltegravir and a second one in phase III clinical development (elvitegravir), genotypic and phenotypic resistance assays are required to guide antiretroviral therapy and to investigate treatment failure. In this study, a genotypic and phenotypic recombinant virus assay was validated for determining resistance against integrase inhibitors. The assays are based on the amplification of a region encompassing not only HIV-1 integrase, but also reverse transcriptase and RNAseH. The overall amplification success was 85% (433/513) and increased to 93% (120/129) for samples with a viral load above 3 log(10) copies/ml. Both B and non-B HIV-1 subtypes could be genotyped successfully (93%; 52/56 and 100%; 49/49, respectively) and reproducibly. The phenotypic assay showed a high success rate (96.5%; 139/144) for subtype B (100%; 19/19) and non-B subtypes (92%; 45/49), and was found to be accurate and reproducible as assessed using well-characterized integrase mutants. Using both assays, baseline resistance to raltegravir and elvitegravir in subtype B and non-B HIV-1 strains selected at random was not observed, although integrase polymorphisms were present at varying prevalence. Biological cutoff values were found to be 2.1 and 2.0 for raltegravir and elvitegravir, respectively. In summary, a genotypic and phenotypic integrase resistance assay was validated successfully for accuracy, reproducibility, analytical and clinical sensitivity, and dynamic range.

Mutations in the thumb-connection and RNase H domain of HIV type-1 reverse transcriptase of antiretroviral treatment-experienced patients.

BACKGROUND: Antiretroviral therapy that targets HIV type-1 (HIV-1) reverse transcriptase (RT) can be linked to mutations in the thumb-connection (amino acids [AA] 241-426) and RNase H (AA 427-560) domains, which could affect drug resistance. METHODS: Genotypical and statistical analyses were performed on HIV-1 RT from 100 antiretroviral treatment-naive and 248 antiretroviral treatment-experienced patients, the majority of whom were infected with HIV-1 subtype B. The RT region was analysed in three parts: the polymerase (AA 1-240), thumb-connection (AA 241-426) and RNase H (AA 427-560) domains. RESULTS: The polymerase domain had statistically significant changes between the two groups at 24 AA positions that are known resistance sites. Within the thumb-connection domain, R284 and N348 had statistically significant changes between the groups (P=0.007 and P< or =0.001, respectively). In treatment-experienced patients, 17.3% had R284K, whereas 24.5% had N348I substitutions. Both R284 and N348 were 100% conserved in treatment-naive patients. Within the RNase H domain, only K451 showed a statistically significant change (P

Small molecule inhibitors targeting HIV-1 reverse transcriptase dimerization.

The enzymatic activities of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are strictly correlated with the dimeric forms of this vital retroviral enzyme. Accordingly, the development of inhibitors targeting the dimerization of RT represents a promising alternative antiviral strategy. Based on mutational studies, we applied a structure-based ligand design approach generating pharmacophoric models of the large subunit connection subdomain to possibly identify small molecules from the ASINEX database, which might interfere with the RT subunit interaction. Docking studies of the selected compounds identified several candidates, which were initially tested in an in vitro subunit association assay. One of these molecules (MAS0) strongly reduced the association of the two RT subunits p51 and p66. Most notably, the compound simultaneously inhibited both the polymerase as well as the RNase H activity of the retroviral enzyme, following preincubation with t(1/2) of about 2 h, indicative of a slow isomerization step. This step most probably represents a shift of the RT dimer equilibrium from an active to an inactive conformation. Taken together, to the best of our knowledge, this study represents the first successful rational screen for a small molecule HIV RT dimerization inhibitor, which may serve as attractive hit compound for the development of novel therapeutic agents.

Apparent defects in processive DNA synthesis, strand transfer, and primer elongation of Met-184 mutants of HIV-1 reverse transcriptase derive solely from a dNTP utilization defect.

The 2',3'-dideoxy-3'-thiacytidine drug-resistant M184I HIV-1 reverse transcriptase (RT) has been shown to synthesize DNA with decreased processivity compared with the wild-type RT. M184A displays an even more severe processivity defect. However, the basis of this decreased processivity has been unclear, and both primer-template binding and dNTP interaction defects have been proposed to account for it. In this study, we show that the altered properties of the M184I and M184A RT mutants that we have measured, including decreased processivity, a slower rate of primer extension, and increased strand transfer activity, can all be explained by a defect in dNTP utilization. These alterations are observed only at low dNTP concentration and vanish as the dNTP concentration is raised. The mutant RTs exhibit a normal dissociation rate from a DNA primer-RNA template while paused during synthesis. Slower than normal synthesis at physiological dNTP concentration, coupled with normal dissociation from the primer-template, results in the lowered processivity. The mutant RTs exhibit normal DNA 3'-end-directed and RNA 5'-end-directed ribonuclease H activity. The reduced rate of DNA synthesis causes an increase in the ratio of ribonuclease H to polymerase activity thereby promoting increased strand transfer. These latter results are consistent with an observed higher rate of recombination by HIV-1 strains with Met-184 mutations.

Efavirenz accelerates HIV-1 reverse transcriptase ribonuclease H cleavage, leading to diminished zidovudine excision.

Previous biochemical studies have demonstrated that synergy between non-nucleoside reverse transcriptase (RT) inhibitors (NNRTI) and nucleoside RT inhibitors (NRTIs) is due to inhibition by the NNRTI of the rate at which HIV-1 RT facilitates ATP-mediated excision of NRTIs from chain-terminated template/primers (T/P). However, these studies did not take into account the possible effects of NNRTI on the ribonuclease H (RNase H) activity of RT, despite recent evidence that suggests an important role for this activity in the NRTI excision phenotype. Accordingly, in this study, we compared the ability of efavirenz to inhibit the incorporation and excision of zidovudine (AZT) by HIV-1 RT using DNA/DNA and RNA/DNA T/Ps that were identical in sequence. Whereas IC(50) values for the inhibition of AZT-triphosphate incorporation by efavirenz were essentially similar for both DNA/DNA and RNA/DNA T/P, a 19-fold difference in IC(50) was observed between the AZT-monophosphate excision reactions, the RNA/DNA T/P substrate being significantly more sensitive to inhibition. Analysis of the RNase H cleavage events generated during ATP-mediated excision reactions demonstrated that efavirenz dramatically increased the rate of appearance of a secondary cleavage product that decreased the T/P duplex length to only 10 nucleotides. Studies designed to delineate the relationship between T/P duplex length and efficiency of AZT excision demonstrated that RT could not efficiently unblock chain-terminated T/P if the RNA/DNA duplex length was less than 12 nucleotides. Taken together, these results highlight an important role for RNase H activity in the NRTI excision phenotype and in the mechanism of synergy between NNRTI and NRTI.