Figure 6
From: Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody
Binding of sdAb19 in Neffin correlates with the inhibition of CD4 internalization. (A) HeLa-CD4 cells were transfected with plasmids for expression of either Nef-GFP or GFP in combination with the plasmid for expression of wild-type or mutated sdAb19 (1:3 Nef:sdAb19 plasmid ratio). Transfected cells were analyzed for CD4 cell surface expression and cell lysates were analyzed by Western blotting (inset panels) as in Figure 3D. (B) HeLa-CD4 cells were transfected with plasmids for expression of either Nef-GFP, mutant Nef-GFP or GFP alone (1:0 plasmid ratio) or in combination with increasing amounts of the plasmid for the expression of wild-type Neffin (1:4 or 1:8 plasmid ratio) and analyzed for CD4 cell surface expression. (C) Model of the complex formation between Nef and Neffin, consisting of the two individual Nef-binding domains sdAb19 and SH3B6. Formation of a stable 2:2 complex leads to coverage of additional surfaces in Nef that increases the inhibitory potential of Neffin against Nef functions.