FIG 2.

Effects of MPER changes in the State 1-stabilized 2-4 RM6 AE Env. (A) The 2-4 RM6 AE Env is an HIV-1_AD8 Env variant in which the pretriggered (State-1) conformation is stabilized, compared to the wild-type HIV-1_AD8 Env (95). Of the 12 changes in the 2–4 RM6 AE Env relative to the wild-type HIV-1_AD8 Env, the three highlighted changes (Q114E, Q567K, and A582T) are the key determinants of the viral phenotypes associated with State-1 stabilization (95). (B, C) The experiments were carried out as described in the legend to Fig. 1. Briefly, cell supernatants containing recombinant luciferase-expressing viruses were collected from transiently transfected HEK293T cells, clarified, and either used directly for infection (left) or incubated with the 19b antibody for 1 h at 37°C (right) before TZM-bl target cells were added. After 48 to 72 h, luciferase activity (RLU) was measured. In the cold sensitivity assay, viruses were incubated on ice for the indicated times before virus infectivity was measured (middle). Note the cold resistance of the 2-4 RM6 AE variants, compared with that of wild-type HIV-1_AD8 in Fig. 1B The results shown are representative of those obtained in two independent experiments, expressed as means and standard deviations from triplicate luciferase readings. The significance of the difference in the cold sensitivity of the 2-4 RM6 AE Env variants was evaluated by a Student’s paired t test.