Mechanistic Analysis of the Broad Antiretroviral Resistance Conferred by HIV-1 Envelope Glycoprotein Mutations

Replication kinetics of Env-A539V in the presence of INSTIs, NRTI, NNRTI, PI, ALLINI, and Ent-Is. The SupT1 T-cell line was transfected with WT or Env-A539V proviral clones in the absence or in the presence of indicated concentrations of ARVs. (A and B) INSTIs; (C and D) NNRTI and NRTI; (E) PI; (F) ALLINI; (G and H) Ent-Is. Virus replication kinetics were monitored by measuring RT activity at the indicated time points. Data are representative of at least two independent experiments. (I) The SupT1 T-cell line was transfected with WT or Env-A539V proviral clones in the absence or in the presence of serial dilutions (0.01 to 3,000 nM) of ARVs. IC₅₀ values were calculated based on RT levels at the peak of virus replication. Fold changes in IC₅₀ were calculated compared to that for the WT. Data from at least two independent experiments are shown as means ± standard errors (SEs). ***, P < 0.001; **, P < 0.01; *, P < 0.05 by unpaired t test. The EFV and NFV data are shown on a separate bar graph to avoid compression of the y axis.

Cell-free infectivity of Env-A539V in the presence or absence of ARVs. (A) RT-normalized virus stocks produced from HeLa cells were used to infect TZM-bl cells. Luciferase activity was measured at 48 h postinfection. Infectivity of WT NL4-3 is normalized to 1.0. (B to I) TZM-bl cells were exposed to 100 TCID₅₀ of WT or Env-A539V virus in the presence of various concentrations (from 0.003 to 3,000 nM) of ARVs and incubated for 48 h. For NFV and BI-224436, 293T cells were transfected with the indicated proviral clones in the presence of the inhibitors. At 48 h posttransfection, the supernatants were used to infect TZM-bl cells. Data from at least three independent experiments are shown as means ± SEs.

Effect of Env-A539V and RT-Y188L mutations on susceptibility to EFV. (A) The SupT1 T-cell line was transfected with the indicated proviral clones in the absence or presence of 1,000 nM EFV. Virus replication kinetics were monitored by measuring RT activity at the indicated time points. Data are representative of three independent experiments. (B) SupT1 T cells were transfected with the indicated proviral clones in the absence or presence of serial dilutions of EFV (0.03 to 1,000 nM). The dose-dependent inhibition curve was determined based on RT values at the peak of multicycle spreading virus replication. (C) TZM-bl cells were exposed to 100 TCID₅₀ of the indicated viruses in the absence or presence of serial dilutions of EFV (0.1 to 10,000 nM). Luciferase activity was measured at 48 h postinfection. (D) IC₅₀ values for EFV in multicycle spreading and cell-free infection based on the data in panels B and C. Data from at least three independent experiments are shown as means ± SEs. Statistical analysis by unpaired t test.

Effect of Env mutations on sCD4-induced and time-dependent gp120 shedding. (A and B) Concentrated viruses were incubated with sCD4 at the indicated concentrations at 37°C for 0 or 2 h. Incubated viruses were subsequently purified through a 20% sucrose cushion, and viral proteins were detected by Western blotting. (A) A representative gel for the sCD4-induced gp120 shedding assay is shown. (B) The ratio of gp120 to p24 was quantified and plotted. Data from at least two independent experiments are shown as means ± SEs. (C) TZM-bl cells were exposed to 100 TCID₅₀ of the indicated viruses incubated with sCD4 prior to infection for 0 or 2 h. Luciferase activity was measured at 48 h postinfection. Data from three independent experiments are shown as means ± SEs. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by unpaired t test, with asterisks for 0 h or 2 h incubation time points indicated in black or gray, respectively. (D and E) Concentrated viruses were incubated at 37°C for the indicated times. Incubated viruses were subsequently purified through a 20% sucrose cushion, and viral proteins were detected by Western blotting. (D) A representative gel for the time-dependent gp120 shedding assay is shown. (E) The ratio of gp120 to p24 was quantified and plotted. Data from at least three independent experiments are shown as means ± SEs. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by unpaired t test.

Sensitivity or binding of Env-A539V to NAbs recognizing different Env conformations, anti-CD4 Ab, and coreceptor antagonist. TZM-bl cells were exposed to 100 TCID₅₀ of WT NL4-3 or Env-A539V viruses in the presence of various concentrations of NAbs (A to J), anti-CD4 Ab (K), and CXCR4 antagonist (L); luciferase activity was measured at 48 h postinfection. Data from three independent experiments are shown as means ± SEs. 293T cells transfected with the green fluorescent protein (GFP)-encoding reporter clone pBR43IeG expressing WT NL4-3 Env or the Env-A539V mutant were preincubated with 17b (M), 10E8 (N), PG16 (O), or PGT145 (P) at 4°C for 1 h. KFS is an Env-defective mutant. The cells were washed, and APC-conjugated anti-human IgG was used to detect bound Ab. APC signals were normalized by GFP signal to calculate the Ab binding efficiency. Data are shown as means ± SEs from three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by unpaired t test or one-way ANOVA and Tukey’s multiple-comparison test.

Selection for DTG resistance with the CCR5-tropic NL(AD8) strain. (A) The SupT1huR5 T-cell line was transfected with pNL(AD8) in the absence or presence of 6.0 nM DTG. At the time point indicated by the arrow, DNA was extracted from the DTG-treated culture and the IN- and Env-coding regions were sequenced, leading to the identification of the Env-N654K mutation. (B) An Env structure of subtype B JR-FL SOSIP.664 (PDB accession number 5FYK [137]), highlighting the location of Env mutations selected in the context of NL4-3 (48) and the Env-N654K mutation selected in NL(AD8). Env amino acid positions are indicated using the NL4-3 numbering. Most of gp120 is shown in white, with gp120 V1/V2 and V3 loops colored in light yellow and light green, respectively. Env-Y61, P81, and A556 are highlighted in cyan; Env-A539 and N654 are highlighted in red and purple, respectively. The structural model was generated using the UCSF Chimera software. (C) The SupT1huR5 T-cell line was transfected with WT or mutant pNL(AD8) in the absence or presence of 300 or 0.3 nM DTG. Replication kinetics were monitored by measuring RT activity at the indicated time points. Data are representative of at least three independent experiments. (D) The SupT1huR5 T-cell line was transfected with WT or mutant pNL(AD8) in the absence or presence of a serial dilution of DTG (1,000 nM to 0.03 nM). DTG IC₅₀ values were calculated based on RT values at the peak of virus replication. Fold changes in IC₅₀ relative to that for WT are indicated. Data from at least three independent experiments are shown as means ± SEs. **, P < 0.01; *, P < 0.05 by unpaired t test. (E) RT-normalized virus stocks produced from HeLa cells were used to infect TZM-bl cells. Luciferase activity was measured at 48 h postinfection. ***, P < 0.001 by unpaired t test. (F) TZM-bl cells were exposed to 100 TCID₅₀ of the indicated viruses in the presence of various concentrations of DTG, and luciferase activity was measured at 48 h postinfection. Data are normalized to WT and are shown as means ± SEs from at least three independent experiments.

Selection for DTG resistance with the subtype C CCR5-tropic isolate K3016. (A) The SupT1huR5 T-cell line was transfected with the K3016 molecular clone in the presence of 3.0 nM DTG, 2.0 nM RPV, or 100 nM FTC. DNA was extracted from the ARV-treated cultures at the peak of replication, and Env-coding regions were sequenced, leading to the identification of the Env-T541I and Env-E621V mutations. Amino acid numbers are based on HXB2 numbering. (B) gp41 HR1 sequences around the K3016 Env-T541I mutation are shown aligned with the NL4-3 sequence. (C to J) The SupT1huR5 T-cell line was transfected with WT K3016 or the indicated Env variants in the absence of DTG, EFV, NFV, and T-20. The supernatants were collected at the indicated time points and were assayed for replication kinetics by measuring RT activity. Data are representative of at three independent experiments. (K) The SupT1huR5 T-cell line was transfected with WT K3016 or the indicated Env variants in the absence or presence of a serial dilution of DTG, EFV, or NFV (0.01 to 300 nM) or T-20 (0.1 to 3,000 nM). IC₅₀s were calculated based on RT values at the peak of virus replication. Fold changes in IC₅₀ relative to that of WT were calculated. (L) RT-normalized virus stocks produced from 293T cells were used to infect TZM-bl cells. Luciferase activity was measured at 48 h postinfection. (M) TZM-bl cells were exposed to 100 TCID₅₀ of the indicated viruses in the presence of a range of DTG concentrations, and luciferase activity was measured at 48 h postinfection. Data are shown as means ± SEs from three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by one-way ANOVA and Tukey’s multiple-comparison test.

The sensitivity of NL(AD8) Env-N654K to NAbs recognizing different Env conformations, anti-CD4 Ab, and entry inhibitors. TZM-bl cells were infected with 100 TCID₅₀ of the indicated viruses in the presence of various concentrations of NAbs (A to G), sCD4 (H), anti-CD4 Ab SIM.4 (I), and entry inhibitors (J to L). Luciferase activity was measured at 48 h postinfection. Data from at least three independent experiments are shown as means ± SEs. ***, P < 0.001; **, P < 0.01; *, P < 0.05 by unpaired t test. The asterisks for 0 h or 2 h incubation with sCD4 (H) are indicated as black or gray, respectively.