Functional Envs lacking CD4 binding do not suppress antigen-induced CD4⁺ T-cell proliferation. 293T cells were transfected with 8X or 8XD Env-encoding mRNA; (A) 8XV3Bal or (B) 8XDV3Bal Env-encoding mRNA, and control poly(AC) RNA and cocultured with CFSE-labeled PBMCs. CD4⁺ T cells were activated with 0.1 μg/mL TSST-1, and proliferation was measured 4 days later using flow cytometry. P values were .01 (A) and less than .005 (B) for comparison to sCD4 treatment. P values were less than .005 in comparing 8XD and 8XDV3Bal with no Env control. Quadrants are labeled with the percent of events in each. (C) Expression of Env protein by mRNA-transfected 293T cells was confirmed by Western blot analysis. (D) Surface expression of functional Env on mRNA-transfected 293T cells was determined using fusion assays. Error bars represent the standard error of the mean. Experiments shown are representative of 3 independent experiments.