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

Abstract

The emergence of HIV-1 drug resistance remains a major obstacle in antiviral therapy. M184I/V and E138K are signature mutations of clinical relevance in HIV-1 reverse transcriptase (RT) for the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and emtricitabine (FTC) and the second-generation (new) nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV), respectively, and the E138K mutation has also been shown to be selected by etravirine in cell culture. The E138K mutation was recently shown to compensate for the low enzyme processivity and viral fitness associated with the M184I/V mutations through enhanced deoxynucleoside triphosphate (dNTP) usage, while the M184I/V mutations compensated for defects in polymerization rates associated with the E138K mutations under conditions of high dNTP concentrations. The M184I mutation was also shown to enhance resistance to RPV and ETR when present together with the E138K mutation. These mutual compensatory effects might also enhance transmission rates of viruses containing these two mutations. Therefore, we performed tissue culture studies to investigate the evolutionary dynamics of these viruses. Through experiments in which E138K-containing viruses were selected with 3TC-FTC and in which M184I/V viruses were selected with ETR, we demonstrated that ETR was able to select for the E138K mutation in viruses containing the M184I/V mutations and that the M184I/V mutations consistently emerged when E138K viruses were selected with 3TC-FTC. We also performed biochemical subunit-selective mutational analyses to investigate the impact of the E138K mutation on RT function and interactions with the M184I mutation. We now show that the E138K mutation decreased rates of polymerization, impaired RNase H activity, and conferred ETR resistance through the p51 subunit of RT, while an enhancement of dNTP usage as a result of the simultaneous presence of both mutations E138K and M184I occurred via both subunits.

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

“…Figure 5 shows that only M184V possessed diminished processivity and that all N348I-containing RTs had processivity similar to that of WT RT, as demonstrated by the band density of full-length (FL) DNA products. The E138K mutant enzyme had higher processivity than did WT RT, consistent with previous results (9,44). These findings show that RTs that contain N348I, i.e., E138K/N348I and E138K/M184V/N348I, do not have diminished enzyme processivity.…”

“…Previous results of steady-state kinetic experiments showed that E138K RT has enhanced dNTP-binding affinity (lower K m value) and can restore the deficit in dNTP usage of RT containing the M184I/V mutations (9,44). To assess the impact of N348I or M184V/N348I on E138K-containing RT on dNTP-binding affinity and catalytic efficiency, steady-state kinetics studies were performed by using WT RT and the E138K, E138K/M184V, and E138K/M184V/N348I RT variants (9,44). The results in Table 4 show that RT enzymes containing E138K/N348I and E138K/M184V/N348I variants had K m values similar to those of WT RT for dTTP but displayed diminished catalytic efficiency (k cat /K m ), i.e., 34% and 39% of the WT value, respectively.…”

“…M184I/V was also common, together with E138K, in patients who failed combination antiretroviral FTC/TDF/RPV therapy in the ECHO and THRIVE clinical studies (36), due to mutual compensatory effects between the E138K and M184I/V substitutions in regard to enzyme processivity, polymerization efficiency, and viral replication capacity (9,43,44,96). A strong association between N348I and M184V has also been observed, and the selection of N348I is associated primarily with the use of ZDV and NVP (8,22,24).…”

“…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 mechanism of E138K-mediated RPV resistance is an increase in the dissociation rate of RPV, which overcomes a smaller enhancement in its rate of association (11). E138K and M184I/V have a mutual compensatory effect in regard to RT processivity and viral replication capacity (9,11,43,44). However, unlike N348I, E138K is relatively rare in treatment-experienced patients (40,(45)(46)(47), despite the fact that it can mutually compensate for M184I/V (43,44).…”