Figure 1.
Schematic display of Nef Interacting (NI) protein design.
(A) Cartoon of interaction motifs within the Nef structure. (B) Protein domains and lipidation signal sequences employed for the design of Nef interacting proteins. The number of different constructs generated and the proposed target sites are shown to the right. (C) Fusion design of four different NI generations. Different recognition domains were assembled in alternating orders and combined with various membrane association motifs to result in different expression constructs, used either for recombinant protein production or in vivo cell expression. (D) Display of the three high affinity binders designed to wrap the interaction surfaces of Nef. (E) SDS PAGE display of selected recombinant Nef and NI proteins used for in vitro binding analyses.
Figure 2.
Isothermal titration calorimetric measurements between Nef and selected Nef interacting proteins.
(A) Binding analyses between Nef and optimized GST-Hck-CD4 chimeras. Displayed are ITC measurements with NI1-1 (Hck, upper left), NI1-2 (Hck-RT loop optimized; upper-right), NI3-1 (CD4-Hck; lower left) and NI3-9 (Hck-CD4; lower right). (B) Contribution of the length of the N-terminal membrane anchor domain of Nef to the binding to NI3-13. (C) Binding between the prototypic Nef inhibitor NI3-9 (HckSH3-polyGly-CD4) and the SH3 domain binding defective Nef (PxxPxR to AxxAxA) mutant. The dissociation constant of 1.2 µM revealed the putative contribution of the dileucine motif of CD4 to the Nef interaction. The thermodynamic parameters and the dissociation constants of the interactions are listed in Table 1.
Table 1.
Thermodynamic parameters of Nef-inhibitor interactions.
Figure 3.
Association of Nef with NIs in human cells.
(A) Localization of Nef and NIs in HeLa cells. GFP or Nef.GFP was co-expressed with an empty control vector or the indicated NIs and subjected to confocal microscopy analysis following fixation and anti-HA immunostaining. Presented are confocal sections of the middle of representative cells. Scale bar = 10 µm. Arrows indicate examples of colocalization between Nef.GFP and NIs. (B) Co-immunprecipitation of Nef.GFP and the indicated NIs from HeLa cells. Shown is a Western blot analysis of Nef.GFP and NIs in the input cell lysate (upper panel) and following anti-GFP immunoprecipitation (lower panel).
Figure 4.
Inhibition of Nef functions by NIs in human cells.
(A) FACS analysis of receptor cell surface density. Depicted are the cell surface levels of the indicated receptors on CHO) hCD4hCCR5 (for CD4 and CCR5) or HeLa-derived TZM (MHC-I, CD71) cells expressing GFP or Nef.GFP together with an empty control plasmid or the indicated NIs. Values are mean +/− STD from triplicate experiments relative to the GFP control sample that was arbitrarily set to 100%. (B) Single round of replication analysis on TZM cells for determination of virion infectivity. 2 ng p24 of HIV-1 wt or ΔNef virions produced in the presence of an empty control plasmid or the indicated NIs were used to infect TZM reporter cells. 36 h post-infection, cells were fixed, stained for β-galactosidase, and the number of blue cells was counted. Data represent mean values +/− STD from triplicate experiments plotted relative to the HIV-1 wt control that was arbitrarily set to 100% (corresponding to 200–300 blue cells/96 well). Asterisks indicate statistical significance as calculated by students t-test (***, p<0.0005; **,p<0.005; *, p<0.05; n.s., non significant). Statistical significance was evaluated between the corresponding GFP and Nef.GFP (A) or HIV-1 wt and ΔNef samples, respectively.
Figure 5.
Structure of the Nef–NI1-2 inhibitor complex.
(A) Ribbon display of NefSF2 (44-210, Δ158-178) in complex with SH3Hck(VSWSPD) (NI1-2). Residues in the complex interface are highlighted. Stacking interactions between F94Nef and W92Hck are shown. (B) Surface display of the NefSF2 core domain structure with the RT-loop sequence of Hck (top) and the newly identified helix α3 (bottom) of Nef shown as ribbon display. The gate keeper residues F94 and W117 of Nef separate two hydrophobic patches in the assembly of the α4-α5 helices that are covered by mutant W92 of Hck and W61 of Nef. (C) Superimposition of the N-terminal regions from the crystal structure determined here and the NMR structure of Nef [61]. (D) NMR mapping experiments revealed displacement of the tryptophane 61 residue by the CD4 part of NI3-13. Shown is a titration series of NI1-2 (left) or NI3-13 (right) to 15N-labeled Nef. Only the spectral region encompassing the HNε resonance signal of tryptophanes is shown.
Table 2.
Crystallographic data collection and structural refinement statistics.