N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

Biondi, Mia J; Beilhartz, Greg L.; McCormick, Susan; Götte, Matthias

doi:10.1074/jbc.m110.105775

Cited by 28 publications

(30 citation statements)

References 51 publications

(26 reference statements)

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“…Our reported increase in the NVP IC 50 of p66 N348I /p51 N348I is consistent with values published elsewhere (2-3-fold increase) (31,57). These in vitro changes are smaller than those observed in cell-based assays for viruses harboring the same mutation (typically 7-8-fold and up to 27-fold) (31)(32)(33)(34).…”

Section: Discussionsupporting

confidence: 77%

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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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Section: Discussionsupporting

confidence: 77%

“…Hence, NVP resistance of CSMs is expected to be affected by RNase H-independent mechanisms and possibly by RNase-dependent mechanisms. It is also possible that resistance is affected by sequence-specific effects on inhibitor susceptibility (57).…”

Section: Discussionmentioning

confidence: 99%

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 connection mutations have been shown to impact DNA polymerization, enhance excision, reduce RNase H activity, and enhance NNRTI resistance 20–22,24,80 . Interestingly, the connection mutations also enhanced the number of hubs and communities in the palm subdomain (Figs.…”

Section: Resultsmentioning

confidence: 99%

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

2013

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HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations.

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“…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%

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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N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

Cited by 28 publications

References 51 publications

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

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

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

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

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