Fig. 2. The NL4-3 7XEnv* variant exhibits high-level DTG resistance in the context of spreading, multi-round infection but not in cell-free, single-round infection.

(A) An infectious molecular clone with seven Env mutations (7XEnv) and a Vpu mutation (denoted by the *) derived from the long-term passaging experiment was constructed by transferring the env amplicon from NL4-3 culture 1 (passage 38, 256 nM DTG; Fig. 1A) into pNL4-3. The locations of mutations in Env are indicated: C1 and C3, first and third conserved domains of gp120, respectively; V2 and V3, second and third variable domains of gp120, respectively; HR1, heptad repeat 1 of gp41; MSD, membrane-spanning domain; CT, cytoplasmic tail. (B) Replication kinetics of the NL4-3 Env variants in the SupT1 T cell line in the absence or presence of DTG. Replication curves obtained in the presence of 0, 3, 100, and 1000 nM DTG are shown. Data are representative of at least three independent experiments. (C) Fold changes in IC₅₀ were calculated compared to that for the WT over a range of DTG concentrations (0.01 to 3000 nM). IC₅₀ values were calculated on the basis of the AUC of the replication kinetics. (D) Single-round, cell-free viral infectivity of the Env variants. Relative infectivity is shown, normalized to 1 for WT NL4-3. (E) DTG sensitivity in the context of cell-free infection. TZM-bl cells were incubated with 100 TCID₅₀ of WT virus or the Env mutants in the presence of various concentrations of DTG. (F) Cell-cell fusion activity of the NL4-3 Env variants. The transfected HEK293T cells were cocultured with TZM-bl cells in the presence of a cocktail of RPV and DTG to prevent productive infection of the TZM-bl cells. Data from at least three independent experiments are shown as means ± SEM. *P < 0.05, unpaired t test.