FIG 3.

Effects of MPER changes in HIV-1_AD8 Envs with State 1-stabilizing changes. The indicated MPER changes were introduced into HIV-1_AD8 Envs containing Q114E/Q567K (A) or Q114E/Q567K/A582T substitutions (B) that have previously been shown to stabilize a pretriggered (State-1) conformation (95). The experiments were carried out as described in the legend to Fig. 1. Briefly, recombinant viruses produced from HEK293T cells transfected with pNL4-3.Luc.R-E- and an Env-expressing plasmid were incubated with TZM-bl target cells for 48 h, after which luciferase activity was measured (left). In the cold sensitivity assay, the virus was incubated on ice before addition to target cells (middle). In the neutralization assay, the virus was preincubated with the 19b antibody for 1 h at 37°C before TZM-bl cells were added to the mixture (right). 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 Env variants was evaluated by a Student’s paired t test.