The Human Immunodeficiency Virus Type 1 Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutation I132M Confers Hypersensitivity to Nucleoside Analogs

Abstract

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a key target for antiretroviral drug development. To date, 12 RT inhibitors have been approved for the treatment of HIV-1 infection and can be classified into two distinct therapeutic groups. These include the nucleoside/nucleotide RT inhibitors (NRTIs) that block HIV-1 replication by acting as chain terminators in DNA synthesis and the nonnucleoside RT inhibitors (NNRTIs) that are allosteric inhibitors of HIV-1 RT DNA polymerization reactions. Although combination therapies that contain two or more RT inhibitors have profoundly reduced morbidity and mortality from HIV-1 infection, their long-term efficacy is limited by the selection of drug-resistant variants of HIV-1. Antiviral drug resistance is defined by the presence of viral mutations that reduce drug susceptibility compared with the drug susceptibilities of wild-type (WT) viruses. Whether or not a particular drug-resistant mutant develops depends on the extent to which virus replication continues during therapy, the ease of acquisition of the particular mutation, and the effect that the mutation has on drug susceptibility and viral fitness. In this regard, we recently detected a novel but rare NNRTI resistance mutation at codon 132 (I132M) in RTs of clinical isolates from patients for whom NNRTI therapy was failing (6, 16). In vitro analyses showed that the I132M mutation in HIV-1 RT conferred high-level resistance to nevirapine and delavirdine (Ͼ10-fold that of the WT) and low-level resistance (ϳ2-to 3-fold that of the WT) to efavirenz (18). In fact, the levels of resistance conferred by I1...

Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations

“…The mutations introduced into the recombinant HIV-1 RT did not interfere with either heterodimer formation or enzyme purification. Consistently with previously published data, the E138K mutation did not have an effect on RT dimerization (1,25).…”

Competitive Fitness Assays Indicate that the E138A Substitution in HIV-1 Reverse Transcriptase Decreases In Vitro Susceptibility to Emtricitabine

“…Of note, we previously reported that an I132M substitution in the ␤7-␤8 loop (that contains residue E138) of the p51 subunit of HIV-1 RT conferred nevirapine resistance but 3TC hypersusceptibility (8). Taken together, these studies suggest that residues in the ␤7-␤8 loop of HIV-1 RT can influence nucleotide selectivity.…”

Molecular Mechanism of Antagonism between the Y181C and E138K Mutations in HIV-1 Reverse Transcriptase

“…Amino acid residue E138 in HIV-1 RT is part of the ␤7-␤8 loop in the p51 subunit, located at the p66/p51 interface, which is a key structural element for RT dimerization, that involves the floor of the NNRTI binding pocket (14,31,32). It was previously shown that E138K did not interfere with either heterodimer formation or enzyme purification (2,29,47). Recombinant WT heterodimeric (p66/p51) RTs and RT enzymes containing each of the E138K, Y181C, and E138K/Y181C substitutions were purified to Ͼ95% homogeneity as demonstrated by Coomassie blue staining of SDS-PAGE gels (Fig.…”