FIG 4.

C-terminal region of 2391 Nef mediates severe impairments in downregulating cell surface SERINC5. CD4⁺ HeLa cells expressing various 2410 and 2391 Nefs with SERINC5.intHA were stained for cell surface SERINC5 and analyzed by flow cytometry. Nef⁺ CD4⁺ HeLa cells were determined by gating on eGFP⁺ cells using an FMO control. (A) Schematic illustrating Nef isolates and C-terminal 2410 Nef chimeras. Chimera break points for each isolate are indicated. Regions belonging to 2391 Nef are indicated with white diagonal stripes, while regions belonging to 2410 Nef are indicated with solid colors. (B) Representative histogram illustrating the cell surface SERINC5 levels on CD4⁺ HeLa cells after gating on single and transfected (eGFP⁺) cells. Geometric mean fluorescence intensities (MFIs) of cell surface SERINC5 are indicated. (C) Summary of the fold downregulation ability (±SE) for cell surface SERINC5. (D) Fold Nef-eGFP expression for each Nef isolate (n = 3). eGFP, enhanced green fluorescent protein; AF647, Alexa Fluor 647; SE, standard error; *, P ≤ 0.05; ****, P ≤ 0.0001; n.s., nonsignificant (P > 0.05).