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

Abstract

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 ...

Subunit-Selective Mutational Analysis and Tissue Culture Evaluations of the Interactions of the E138K and M184I Mutations in HIV-1 Reverse Transcriptase

“…Recently, subunitspecific analyses showed that the decreased RNase H activity conferred by the N348I mutation mapped to the p51 subunit (44,57). We have now extended our previous results (54) to show that the E138 residue in the ␤7-␤8 loop of the p51 subunit also plays a key role in regulating the RNase H processing activity.…”

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

“…Substitutions at certain residues in HIV-1 RT can impair RNase H activity and contribute to reductions in HIV-1 replication fitness (69,70). 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).…”

“…For example, the M184V mutation diminishes dNTP usage and possesses a deficit in enzyme processivity, which can be correlated with lower replication fitness in cell types that have small dNTP pools (78,82). However, enzyme processivity is not always directly proportional to viral replication capacity, and both E138K and N348I have been demonstrated to compensate for the deficit in processivity associated with M184V (8,26,44). To investigate whether interactions between N348I or M184V/N348I and E138K might have an impact on enzyme processivity, we performed gel-based single-cycle processivity assays using recombinant WT RT or RT containing the E138K, M184V, E138K/N348I, and E138K/M184V/N348I substitutions at low dNTP concentrations.…”

“…The polymerase domain consists of the finger, palm, and thumb subdomains, which are analogous to a right hand in shape (7). Subunit-specific mutational analyses have shown that p51 is involved in the fine-tuning of RT enzymatic activities (8)(9)(10)(11).…”

“…The N348I mutation emerged early in ZDV-and ddIcontaining antiretroviral therapy and frequently coappears with TAMs, M184V, and the NNRTI RAMs K103N, Y181C/I and G190A/S (19,22,24,26,(28)(29)(30). N348I is especially associated with the simultaneous use of ZDV and NVP (8,22,24) and was also five times more prevalent in patients receiving both ZDV and 3TC than in those receiving ZDV alone (26).…”