A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

Nikolenko, Galina N.; Delviks-Frankenberry, Krista A.; Pathak, Vinay K.

doi:10.1128/jvi.01545-09

Cited by 42 publications

(50 citation statements)

References 59 publications

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“…In addition, some NNRTI resistance mutations are associated with impaired RNase H activity (69,(71)(72)(73)(74)(75), and N348I has been shown to reduce the rate of RNA template degradation (8,10,24,76). Furthermore, reduced RNase H activity may be associated with enhanced NNRTI resistance (77). To investigate the impact of N348I or M184V/N348I on the RNase H activity of E138K-containing RT, we monitored multicycle RNase H-mediated RNA cleavage in time course experiments using WT RT and the E138K, E138K/M184V, and E138K/ M184V/N348I RT variants.…”

Section: Resultsmentioning

confidence: 99%

“…Transit-kinetic analysis has shown that E138K resistance to RPV is due mainly to an enhanced dissociation rate of RPV (11). N348I also confers resistance to NVP through decreased binding affinity (8) while inhibiting RNase H activity (10,77,99). Further biochemical and structural analyses may lead to the design of better NNRTIs with improved resistance profiles and potency.…”

Section: Discussionmentioning

confidence: 99%

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The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity

Colby-Germinario

Oliveira

et al. 2014

J Virol

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Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/ M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2-and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity

Colby-Germinario

Oliveira

et al. 2014

J Virol

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“…Such changes may not significantly alter the K d-DNA but could affect proper alignment of DNA with dNTP substrates or stability of the catalytic complex. The enhanced processivity of N348I RT may also contribute to NRTI resistance as it is possible that this property could provide more opportunities for unblocking chainterminated primers or NNRTI dissociation (38,39). The molecular details of these effects are not yet fully understood and should be addressed by structural studies of N348I RT.…”

Section: Discussionmentioning

confidence: 99%

“…Less clear is the role of RNase H in NVP resistance. Nikolenko et al (38) proposed that the decrease in RNase H activity of CSMs preserves the RNA template and provides more time for NNRTIs to dissociate from the RT, resulting in the resumption of DNA synthesis and FIGURE 9. A, superposition of molecular models of WT RT (multicolored schematic; see below) and p66 N348I / p51 N348I RT (magenta schematic).…”

Section: Discussionmentioning

confidence: 99%

“…Nikolenko et al (30,38) proposed that a number of CSMs increase AZT resistance by reducing template RNA degradation, thereby preserving the RNA template and providing additional time for RT to excise AZT monophosphate. Yap et al (31) showed that N348I reduces the rate of RNA template degradation by RT in either a WT background or in the presence of excision-enhancing AZT resistance mutations.…”

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confidence: 99%

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The N348I Mutation at the Connection Subdomain of HIV-1 Reverse Transcriptase Decreases Binding to Nevirapine

Schuckmann

Marchand

Hachiya

et al. 2010

Journal of Biological Chemistry

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The N348I mutation at the connection subdomain of HIV-1 reverse transcriptase (RT) confers clinically significant resistance to both nucleoside and non-nucleoside RT inhibitors (NNRTIs) by mechanisms that are not well understood. We used transient kinetics to characterize the enzymatic properties of N348I RT and determine the biochemical mechanism of resistance to the NNRTI nevirapine (NVP). We demonstrate that changes distant from the NNRTI binding pocket decrease inhibitor binding (increase K d-NVP ) by primarily decreasing the association rate of the inhibitor (k on-NVP ). We characterized RTs mutated in either p66 (p66 N348I /p51 WT ), p51 (p66 WT /p51 N348I ), or both subunits (p66 N348I /p51 N348I ). Mutation in either subunit caused NVP resistance during RNA-dependent and DNA-dependent DNA polymerization. Mutation in p66 alone (p66 N348I / p51 WT ) caused NVP resistance without significantly affecting RNase H activity, whereas mutation in p51 caused NVP resistance and impaired RNase H, demonstrating that NVP resistance may occur independently from defects in RNase H function. Mutation in either subunit improved affinity for nucleic acid and enhanced processivity of DNA synthesis. Surprisingly, mutation in either subunit decreased catalytic rates (k pol ) of p66 N348I /p51 N348I , p66 N348I /p51 WT , and p66 WT /p51 N348I without significantly affecting affinity for deoxynucleotide substrate (K d-dNTP ). Hence, in addition to providing structural integrity for the heterodimer, p51 is critical for fine tuning catalytic turnover, RNase H processing, and drug resistance. In conclusion, connection subdomain mutation N348I decreases catalytic efficiency and causes in vitro resistance to NVP by decreasing inhibitor binding.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) converts the viral single-stranded, positive sense RNA genome to a double-stranded DNA, which is integrated into the host genome. To achieve this task, RT possesses multiple enzymatic activities, including both DNA-dependent and RNA-dependent DNA polymerase activities and RNase H activity. RT is a heterodimer composed of 66-kDa (p66) and 51-kDa (p51) subunits. p66 is 560 amino acids long and comprises the spatially distinct polymerase and RNase H domains. The polymerase domain of p66 includes the fingers, palm, and thumb subdomains that resemble a clasping right hand connected to the RNase H domain through the connection subdomain. p51 contains the first 440 amino acids of p66 and is derived by HIV-1 protease-mediated cleavage of an RNase H domain from the p66/p66 homodimer. Because p51 of the heterodimer has no enzymatic function, it has been proposed that its role is simply to provide structural support to p66.HIV-1 RT has been a prominent target of anti-AIDS therapies. There are two classes of approved drugs that target RT: the nucleoside RT inhibitors (NRTIs) 3 and the non-nucleoside RT inhibitors (NNRTIs). NRTIs mimic deoxynucleotide triphosphate (dNTP) substrates required for DNA synthesis. Once integrated into the ...

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Development and validation of a high performance liquid chromatography method for determination of 6‐benzyl‐1‐benzyloxymethyl‐5‐iodouracil (W‐1), a novel non‐nucleoside reverse transcriptase inhibitor and its application to a pharmacokinetic study in rats

Wang

et al. 2015

Biomedical Chromatography

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A sensitive and selective high-performance liquid chromatographic (HPLC) method for determination of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor in rat plasma, was developed and validated. Chromatographic separation of W-1 and megestrol acetate (internal standard) was achieved on a reversed-phase C18 column at 25°C. The mobile phase was consisted of acetonitrile-water (60:40, v/v) and pumped at a flow rate of 1.0 mL/min. The ultraviolet (UV) detector was set at the absorption wavelength of 284 nm. The calibration curve for W-1 was linear over the concentration range of 0.01-8 µg/mL and the lower limit of quantification was 10 ng/mL. The intra- and inter-day precision and accuracy were <8.9 and 5.3%, respectively. The extraction recoveries ranged from 97.9 to 101.6%. The validated HPLC method was successfully applied to a pharmacokinetic study of W-1 in rats.

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A Novel Molecular Mechanism of Dual Resistance to Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitors

Cited by 42 publications

References 59 publications

The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity

The Connection Domain Mutation N348I in HIV-1 Reverse Transcriptase Enhances Resistance to Etravirine and Rilpivirine but Restricts the Emergence of the E138K Resistance Mutation by Diminishing Viral Replication Capacity

The N348I Mutation at the Connection Subdomain of HIV-1 Reverse Transcriptase Decreases Binding to Nevirapine

Development and validation of a high performance liquid chromatography method for determination of 6‐benzyl‐1‐benzyloxymethyl‐5‐iodouracil (W‐1), a novel non‐nucleoside reverse transcriptase inhibitor and its application to a pharmacokinetic study in rats

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