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.

Fig 1

Purification of recombinant HIV-1 RTs. (A) Coomassie brilliant blue staining of purified heterodimer RTs following 8% SDS-PAGE. The purification of heterodimeric RT enzymes was achieved by the attachment of a His₆ tag at the N terminus of the p51 subunit through immobilized metal affinity chromatography (IMAC). MW, molecular weight standards (in thousands). Lanes: 1, p66_WT/p51_WT; 2, p66_E138K/p51_E138K; 3, p66_WT/p51_E138K; 4, p66_E138K/p51_WT; 5, p66_E138K+M184I/p51_WT; 6, p66_E138K+M184I/p51_E138K; 7, p66_M184I/p51_E138K; 8, p66_WT/p51_M184I. The positions of purified recombinant RT heterodimers are indicated on the right. (B) Comparison of specific activities of recombinant subunit-selective mutant RT enzymes relative to that of the WT enzyme. DNA polymerase activity was assessed as described in Materials and Methods. Data from a representative experiment performed in triplicate are shown as means ± standard deviations.

Fig 2

Subunit-specific analysis of the effect of the E138K mutation in HIV-1 RT on RNase H activity. (A) Graphic representation of the substrate RNA-DNA (kim40R/kim32D) duplex used to monitor the cleavage efficiency of recombinant RT. The 40-mer RNA kim40R was labeled at its 5′ terminus with ³²P and annealed to a 32-mer DNA oligonucleotide, kim32D. The positions of cleaved products are indicated by arrows. (B) RNase H activities were analyzed by monitoring substrate cleavage in time course experiments in the presence of a heparin trap. The positions of cleaved products are indicated on the left. The uncleaved substrate indicates the variability in RNase H activity among different RT enzymes. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis.

Fig 3

Time course experiments showing the subunit-specific effects of the E138K mutation in HIV-1 RT on rates of DNA polymerization. The ³²P-labeled D25 primer (³²P-D25) was annealed to the 497-nt RNA template, and extension assays were performed with an excess of recombinant RT enzymes at dNTP concentrations of 200 μM. Reactions were stopped at 30 s (30″) and 60 s (60″). The sizes of some fragments of the ³²P-labeled 25-bp DNA ladder (Invitrogen) in nucleotide bases are indicated on the left. The longest extension products generated at 60 s are identified by arrows and indicate differences in polymerization rates.

Fig 4

The E138K mutation in HIV-1 RT restores enzyme processivity of the M184I enzyme at low dNTP concentrations via both subunits. The processivity of purified recombinant RT enzymes was analyzed by using a 5′-end-labeled DNA primer (D25) annealed to a 471-nt RNA template as the substrate; the resulting full-length DNA (FL DNA) is 471 nt in size. Processivities were determined by the size distribution of DNA products in fixed-time experiments at low concentrations of dNTPs (0.5 μM) in the presence of a heparin trap. The sizes of some fragments of the ³²P-labeled 25-bp DNA ladder (Invitrogen) in nucleotide bases are indicated on the left. All reaction products were resolved by denaturing 6% polyacrylamide gel electrophoresis and visualized by phosphorimaging. Positions of the ³²P-labeled D25 primer (³²P-D25) and the 471-nt full-length extension DNA (FL DNA) product are indicated on the right.