The M230L nonnucleoside reverse transcriptase inhibitor resistance mutation in HIV-1 reverse transcriptase impairs enzymatic function and viral replicative capacity

Abstract

The M230L mutation in HIV-1 reverse transcriptase (RT) is associated with resistance to first-generation nonnucleoside reverse transcriptase inhibitors (NNRTIs). The present study was designed to determine the effects of M230L on enzyme function, viral replication capacity (RC), and the extent to which M230L might confer resistance to the second-generation NNRTI etravirine (ETR) as well as to the first-generation NNRTIs efavirenz (EFV) and nevirapine (NVP). Phenotyping assays with TZM-bl cells confirmed that M230L conferred various degrees of resistance to each of the NNRTIs tested. Recombinant viruses containing M230L displayed an 8-fold decrease in RC compared to that of the parental wild-type (WT) virus. Recombinant HIV-1 WT and M230L mutant RT enzymes were purified; and both biochemical and cell-based phenotypic assays confirmed that M230L conferred resistance to each of EFV, NVP, and ETR. RT that contained M230L was also deficient in regard to each of minus-strand DNA synthesis, both DNA- and RNA-dependent polymerase activities, processivity, and RNase H activity, suggesting that this mutation contributes to diminished viral replication kinetics.

FIG. 1.

Inhibition of DNA-dependent DNA polymerase RT activity by NNRTIs determined by a fixed-time gel-based RT assay. (A) Graphic representation of the primer-template system (ppt17D-ppt57D) used to monitor the inhibition of HIV-1 RT DNA polymerase activity by NNRTIs. The 17-mer DNA primer ppt17D was labeled with ³²P at the 5′ terminus and annealed to the 57-mer DNA template ppt57D. +1 and +4, positions of the first and the last nucleotides incorporated, respectively; *, position of the incorporated ddGTP. (B) Dose-dependent inhibition of DNA polymerase activity by NNRTIs. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis, visualized by phosphorimaging, and quantified with ImageQuant software (GE Healthcare). The positions of the labeled primer (P) and the full-length extension product (+4) are indicated on the left. The concentrations of the NNRTIs used are as follows: for ETR, 0, 0.6, 1.7, 5, 15.2, 45.6, 137, and 411 nM and 1.23, 3.7, and 11.1 μM; for DAP, 0, 5, 15, 45.7, 137, and 410 nM; 1.23, 3.7, 11.1, 100, and 500 μM; and 1 mM; for EFV, 0, 0.56, 1.69, 5, 15, 45.7, 137, and 410 nM and 1.23, 3.7, 11.1, 33.3, and 100 μM; and for NVP, 0, 10, 31, 95, 285, 857 nM; 2.5, 7.7, 23, 69, 208, and 625 μM; and 1.87 mM.

FIG. 2.

Effect of the M230L mutation on viral replicative capacity. Virus stocks were prepared through transfection of HEK 293T cells with proviral clones pNL4-3_WT and pNL4-3_M230L, normalized for RT activity, and used to infect TZM-bl cells to monitor viral replication. Luciferase activity was measured at 48 h postinfection. The relative infectivities of the WT and M230L viruses are shown on the y axis, while the x axis denotes the input virus stocks expressed as RT activity. These values translate to an 8-fold virus replication disadvantage for HIV-1_M230L compared with the replication capacity of HIV-1_WT.

FIG. 3.

Efficiency of tRNA₃^Lys-primed minus-strand ssDNA synthesis in cell-free assays. The efficiencies of synthesis of full-length DNA with WT and M230L mutant RTs were compared in time course experiments. (A) Graphic representation of the cell-free system used for the synthesis of full-length minus-strand ssDNA. The PBS RNA template used in this system consists of 258 nt at the 5′ end of the HIV-1 genome, which contains the R, U5, and PBS regions. (B) Synthesis of full-length (FL) DNA by recombinant WT and mutant enzymes monitored in time course experiments. The reactions were initiated by the addition of 10 μM dNTPs and were monitored by measurement of the incorporation of [α-³²P]dCMP into the extending DNA strand. The reactions were stopped at different time points during a period of 45 min. Time point zero indicates samples taken at 20 s after initiation. The full-length DNA product and the +1, +3, and +5 pausing sites are shown on the right.

FIG. 4.

Processive polymerization by WT and M230L mutant RTs. (A) Graphic representation of the T-P systems used to monitor the DDDP and RDDP activities of recombinant RTs. DNA primers ppt19D and kim17D were labeled with ³²P and annealed to DNA template ppt57D and RNA template kim40R, respectively. (B) Polymerization activities were analyzed by monitoring the percentage of full-length (FL) DNA synthesis in time course experiments in the presence of the heparin trap. The position of full-length DNA is indicated on the left side of the panel. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis, visualized by phosphorimaging, and quantified with ImageQuant software (GE Healthcare). (C) Graphic representation of the calculated percentage of full-length DNA synthesis in the time course experiments from the gel results. Band intensities were calculated by ImageQuant software, and the data were curve fit by GraphPad Prism (version 5.01) software.

FIG. 5.

Reduced polymerase processivity of RT containing the M230L mutation. The processivities of the recombinant WT and M230L RT proteins were assessed with a homopolymeric RNA template poly(rA)-oligo (dT)_12-18 DNA primer. The DNA primer was labeled with ³²P at the 5′ terminus and was annealed to the poly(rA) RNA template at an equimolar ratio. Processivities were analyzed by monitoring the size distribution of the DNA products in fixed-time experiments in the presence of a heparin trap. Parallel reactions were run in the absence of the heparin trap to ensure that similar amounts of enzyme activities were present in the reactions. Lane C, control reaction to verify the efficiency of the heparin trap by preincubation with substrate prior to addition of RT; lane M, a 25-bp DNA ladder used as a size standard. The sizes (in kilobases) of some fragments of the standard are indicated on the right. All reaction products were resolved by denaturing 6% polyacrylamide gel electrophoresis and were visualized by phosphorimaging.

FIG. 6.

RNase H activities of WT and M230L recombinant RTs. (A) Graphic representation of the substrate RNA-DNA (kim40R-kim32D) duplex used to monitor the cleavage efficiency of the M230L and WT RTs. The 40-mer RNA kim40R was labeled at its 5′ terminus with ³²P and annealed to the 32-mer DNA oligonucleotide kim32D. (B) RNase H activities were analyzed by monitoring substrate cleavage in time course experiments in the absence (left panel) or the presence (right panel) of a heparin trap. The positions of the cleaved products are indicated on the left side. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis.