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

Abstract

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.

Impact of Drug Resistance-Associated Amino Acid Changes in HIV-1 Subtype C on Susceptibility to Newer Nonnucleoside Reverse Transcriptase Inhibitors

“…When it occurs in combination with other NNRTI resistance-associated amino acid substitutions, such as K103N and Y181C, N348I has been shown to contribute to EFV, ETR, and RPV resistance (59,60). In addition, RPV resistance is enhanced by this substitution in the background of E138K (61). We have confirmed in HIV-1 subtype C that N348I alone does not affect ETR/RPV susceptibility but can significantly reduce ETR and/or RPV susceptibility when it occurs in combination with other NNRTI resistance-associated substitutions, particularly Y181C.…”

Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

“…In contrast, M184I does not result in a defect in the efficiency of polymerization at high dNTP concentrations (29,42), but the simultaneous presence of M184I and E138K can restore the efficiency of processive DNA synthesis and viral replication capacity (29). Another example is the N348I substitution in the connection subdomain of HIV-1 RT, which significantly decreases catalytic efficiency (43) and diminishes polymerization efficiency without impairing enzyme processivity, as demonstrated in gel-based assays (30). To determine whether W153L might affect the efficiency of DNA polymerization, we ran gel-based RNA-dependent DNA polymerase assays at high dNTP concentrations (200 M) in time course experiments using the WT, W153L, W153L/K65R, W153L/M184I, W153L/K101E, W153L/K103N, W153L/E138K, and W153L/Y181C recombinant RT enzymes.…”

“…RNase H activity was assayed using a 41-mer 5=-end 32 P-labeled heteropolymeric RNA template termed kim40R that was annealed to a complementary 32-nucleotide DNA oligomer termed kim32D at a 1:4 molar ratio as described previously (29,30). The reactions were conducted at 37°C in mixtures containing an RNA-DNA duplex substrate (20 nM) with equal amounts of RT enzymes in assay buffer (50 mM Tris-HCl, pH 7.8, 60 mM KCl, and 5 mM MgCl 2 ).…”

Identification of Novel Resistance-Related Polymorphisms in HIV-1 Subtype C RT Connection and RNase H Domains from Patients Under Virological Failure in Brazil

“…34 The N348I mutation occurred in 11% of viruses in Southern Brazil and it is associated with major resistance to nevirapine, etravirine, and rilpivirine, and as an accessory mutation for thymidine analogs. 40,41,51,52 Of note, A376S has been associated with failure to NNRTI, particularly nevirapine-containing regimens, 53,54 suggesting a functional link between this mutation and K103N.…”