FIG 6.

Mutations of 2410 Nef M₁₇₀ or H₁₇₃ do not impact SERINC5 downregulation. CD4⁺ HeLa cells expressing 2410 and 2391 Nef as well as the respective 2410 Nef mutants with SERINC5.intHA were stained for cell surface CD4 and SERINC5 and analyzed by flow cytometry. Nef⁺-transfected CD4⁺ HeLa cells were determined by gating on eGFP⁺ cells using an unstained control. (A) Amino acid sequence alignment comparing residues 151 to 174 of 2410 and 2391 Nef. Boldfaced residues indicate polymorphisms between the two Nef isolates within this region. The central flexible loop and Nef dileucine motif are denoted by purple and red boxes, respectively. (B) Schematic illustrating the Nef isolates and 2410 Nef chimeras/mutants utilized. Chimera break points for each isolate are indicated accordingly. Regions belonging to 2391 Nef are indicated with white diagonal stripes, while regions belonging to 2410 Nef are indicated with solid colors. (C and D) Representative histograms illustrating cell surface SERINC5 (C) and CD4 (D) levels on CD4⁺ HeLa cells after gating on single and transfected (eGFP⁺) cells. Geometric mean fluorescence intensities (MFIs) of the respective cell surface proteins are indicated. (E to G) Summary of the fold downregulation ability (±SE) for cell surface SERINC5 (E), cell surface CD4 (F), and fold Nef-eGFP (G) expression for each Nef isolate (n = 3). eGFP, enhanced green fluorescent protein; AF647, Alexa Fluor 647; PE, phycoerythrin; SE, standard error; *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; n.s., nonsignificant (P > 0.05).