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

Abstract

We characterized the relative fitness of multiple nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistant HIV-1 variants in the presence of etravirine (ETV), rilpivirine (RPV), and/or the nucleoside RT inhibitor emtricitabine (FTC) by simultaneous competitive culture and 454 deep sequencing. The E138A substitution, typically associated with decreased virologic responses to ETV-and RPV-containing regimens, confers a clear fitness advantage to the virus in the presence of FTC and decreases FTC susceptibility 4.7-fold. Complera is a fixed-dose antiviral drug combination used as a first-line antiretroviral therapy regimen for the treatment of HIV-1 infection. Complera is composed of the nucleoside reverse transcriptase (RT) inhibitors (NRTIs) emtricitabine (FTC) and tenofovir disoproxil fumarate and the nonnucleoside RT inhibitor (NNRTI) rilpivirine (RPV). In the phase III ECHO and THRIVE clinical trials, the most frequent substitution combination that emerged in the RT gene of Complera virologic failures was E138K and M184I (1, 2). In this regard, Hu and Kuritzkes (3) and Xu et al. (4) reported that the E138K substitution compensated for the poor replicative capacity of M184I. However, Kulkarni et al. found that HIV-1 containing E138K and M184I was less fit than viruses containing either E138K or M184I (5). In addition to E138K, the L100I, K101E/P, E138A/G/Q, Y181C/I/V, Y188L, G190A/S/E, and M230L substitutions are associated with RPV resistance.Recently, we developed an efficient method to characterize the relative fitness of multiple HIV-1 mutants simultaneously (6). Briefly, we generated 15 infectious viruses (HIV-1 LAI ) containing the single NNRTI resistance substitutions V90I, K101P, K103N, V108I, E138A, E138K, V179D, Y181C, Y181I, Y181V, Y188C, G190A, G190S, M230L, and P236L. A wild-type (WT) virus and the 15 mutant viruses were then normalized for relative infectivity (infectious units/ng p24), pooled, and used to infect 1.6 ϫ 10 6 HUT-78 cells at a multiplicity of infection of 0.004. Culture supernatants were collected at 3-day intervals, viral RNA was extracted, and deep sequencing (454 GS Junior) of an amplicon spanning RT codons 96 to 194 was used to quantify the distribution of mutants at each time point. Using this method, we previously showed that the Y181V substitution in the HIV-1 RT confers a selective advantage to the virus over the 14 other NNRTI resistance substitutions in the presence of the NNRTI etravirine (ETV; Fig. 1B) (6). We next asked whether the fitness landscape was altered if FTC was combined with ETV. In the presence of 200 nM ETV and 10 M FTC, longitudinal deep sequencing revealed that the Y181V virus (Fig. 1C). Unexpectedly, we found that in experiments carried out in the presence of 10 M FTC (5ϫ to 10ϫ the EC 50 ), the E138A HIV-1 exhibited a clear fitness advantage (Fig. 1D). The frequency of the E138A variant increased from 9.6% at day 0 to 28% by day 6. The frequencies of HIV-1 containing the V179D or E138K substitutions were also increased, whereas all of the o...

Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase

“…Conversely, E138Q conferred a 4.3-fold EC 50 increase above WT HIV-1 (2.85 Ϯ 0.08 nM) and no difference from WT RT-SHIV (0.30 Ϯ 0.05 nM). These EC 50 s for WT, E138G, and E138Q HIV-1 were similar to values previously reported (55,57).…”

Long-Acting Rilpivirine (RPV) Preexposure Prophylaxis Does Not Inhibit Vaginal Transmission of RPV-Resistant HIV-1 or Select for High-Frequency Drug Resistance in Humanized Mice

“…In this study, classic RPV-associated resistance mutations (K101E, E138K, and E138Q) arose at low frequency in a subset of both RPV LA-treated animals and untreated animals. However, these mutations arise from a single base change and confer only 2-to 5-fold resistance to RPV, according to our data and those of others (25,58,60,65,68,79). It is possible that some mutations selected in genomes already encoding Y181V lead to additive levels of resistance, but this is unlikely to have much effect in a virus that already has Ͼ20-fold reduced susceptibility to RPV and that can be transmitted despite high concentrations of drug in tissues and blood.…”

Rilpivirine resistance and the dangerous liaisons with substitutions at position 184 among patients infected with HIV-1: Analysis from a national drug-resistance database (ARCA)

“…Recent evidence from the SPREAD program pointed out that E138A (which is included in the IAS‐USA mutation list) is the most frequent NNRTI mutation in naive patients [Hofstra et al, ]. Also, in vitro experiments showed that E138A is most common in HIV‐1 subtype C than B [Sluis‐Cremer et al, ] and that this mutation decreases in vitro susceptibility to 3TC/FTC and RVP [Sluis‐Cremer et al, ].…”