Fig 1.
Hck-activating potential of selected HIV-1 Nefs.
(A) Hck-expressing HZ-1 cells were co-transfected with vectors for paxillin and Nef from the indicated HIV-1 group M, N, O or P clones, or mutants thereof. Lysates of the transfected cells were analyzed by Western blotting using antibodies against paxillin (PXN), phosphorylated paxillin (pPXN) and Nef. (B) HZ-1 cells were transfected with an AP-1 transcription factor-driven luciferase reporter alone (Control) or together with the indicated Nefs variants. Luciferase activity was measured in cells harvested 24 h post-transfection, and normalized to the corresponding control sample that was set to 1. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid at position 120 of each Nef protein is indicated, aromatic residues are shown as a black 1-letter symbol in a white sphere, and isoleucine as a white letter in a black sphere. (C) Jurkat T cells stably expressing CD4 were infected with lentiviral vectors expressing GFP alone (No Nef) or together with plasmids expressing the indicated Nefs. CD4 down-regulation by Nef was measured using flow cytometry 48 h after lentiviral transduction, and histograms illustrating cell surface levels of CD4 among the GFP positive Jurkat cells are shown. Original dot plots are shown in S1 Fig.
Fig 2.
HIV-1 M consensus Nef amino acid sequence (A) and an alignment of the conserved central region (underlined in A) of selected Nef proteins from different primate lentiviruses (B). The key residues of the core SH3 docking site (PxxPxR motif), including R77, are highlighted in light blue. Residues involved in a binding pocket for the Hck SH3 domain RT-loop that are conserved in HIV-1 and HIV-1-like Nef proteins are highlighted in yellow. Residues at positions 83 and 120 forming the R-clamp coordinating the positioning of R77 are shown in bold and colored according to their side chain properties (small in green, hydrophilic in blue, aliphatic or methionine in red, aromatic in brown). The names of HIV/SIV strains from which Nef proteins were included in this study are shown on the left.
Fig 3.
(A) Structure of the HIV-1 Nef (green)–SH3 (pink) complex (PDB ID 1EFN) highlighting critical residues in the interaction interface. (B) Close up view of the R clamp, stabilized by stacked Y-R-W side chains and a salt bridge between R77 (Nef) and D17 (SH3) side chains. (C) In the Y120I structure, one side of the R77 guanidinium plane is lacking non-bonded interactions. (D) In the A83M/Y120I, structure the long methionine side chain re-establishes non-bonded contacts for R77. The presented mutated complex structures were created by replacing Y120 or Y120 and A83 in UCSF Chimera [45] and minimizing the structures with AMBER [46].
Fig 4.
Role of residue 83/120 pairing in the HIV-1 N Nef R-clamp.
(A) HZ-1 cells were transfected with an AP-1-dependent luciferase reporter alone (Control) or together with wild type (WT) versions of YBF30 or 2693BA Nef, or mutants thereof carrying reciprocal amino acid changes of residue 83 (YBF30 Q83M and 2693BA M83Q). Luciferase activity was measured from cells 4 h post-transfection, and normalized to the corresponding control sample that was set to one. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid combinations at positions 83 and 120 are indicated here and in the figures below as single-letter symbols and color-coded according to the residue classification shown in Table 1. Specifically, small (G or A) or hydrophilic residues (D, E, Q, or S) at position 83 are shown as a black 1-letter symbol in a white sphere (here Q83 in YBF30), whereas aliphatic or methionine residues at this position are shown as a white letter in a black sphere (here M83 in 2693BA). At position 120, aromatic residues (F or Y) are shown as a black letter in a white sphere (not found in YBF30 or 2693BA), whereas isoleucine is shown as a white letter in a black sphere (here I120 in YBF30 and 2693BA). (B) CD4 downregulation by Nef in stably transduced Jurkat T cells was examined as in Fig 1C. Original dot plots are shown in S2 Fig.
Table 1.
R-clamp residue combinations in the HIV-1-like superfamily of Nef proteins.
Fig 5.
R-clamp architecture of SIVcpz Nef proteins.
(A) HZ-1 cells were transfected with an AP-1-luciferase reporter alone (Control) or together with wild-type or the indicated mutants of Nef from SIV strains from Pan troglodytes troglodytes (MB897, Cam5, and EK505) or Pan troglodytes schweinfurthii (Nok5 and Tan2). Luciferase activity in the transfected lysates was analyzed as in Fig 1C. The data shown are derived from three independent experiments, with SE indicated by error bars. The amino acid combinations at positions 83 and 120 are indicated as single-letter symbols and color-coded as explained in Fig 4. (B) CD4 downregulation by Nef in stably transduced Jurkat T cells was examined as in Fig 1C. Original dot plots are shown in S3 Fig.
Fig 6.
Comprehensive testing of R-clamp residue pairing rules by mutagenesis of SF2-Nef.
Nef-induced AP-1 activity in cells transfected without Nef (Control) or with the indicated wild-type or mutant versions of SF2 Nef was analyzed as in Fig 1B. The amino acid combinations at positions 83 and 120 of these SF2 Nef variants are indicated as single-letter symbols that are color-coded as explained in Fig 4.
Fig 7.
Role of R-clamp residues 83/120 in intracellular Hck-Nef complex formation.
HEK293 cells were transfected with Myc-tagged wild-type HIV-1 M SF2 Nef (WT) or its mutants including Y120I, A83L-Y120I, P76A-P78A (AxxA), A83Q, and A83Q-Y120I) together with biotin acceptor domain-tagged wild-type Hck-p59 (WT) (A.) or its SH3 mutant Hck-C3 (B.). The Nef-contacting RT-loop residues centered around the critical SH3 amino acid 13 (Ile in WT and Asp in C3) are shown on top of these figures. Lysates of transfected cells were subjected to anti-Myc immunoprecipitation followed by Western blotting analysis of the immune complexes using labeled streptavidin (top panels). Equal Hck and Nef expression in the total lysates was confirmed by labeled streptavidin (Hck) (middle panels) or an anti-Myc (Nef) antibody (bottom panels).
Fig 8.
Role of the R-clamp in activation of in vitro differentiated macrophages by Nef.
The indicated wild-type or mutant Nef proteins from HIV-1 M (panels A. and B.), HIV 1 N (panel A.), or SIVcpz(P.t.s) (panel A.) were expressed via lentiviral transduction in M1-like macrophages differentiated from THP-1 cells, followed by Western blotting analysis to compare the expression of Nef, total ERK1/2, and phosphorylated ERK1/2 in these cells or control macrophages transduced with an empty lentivector (-).
Fig 9.
Putative cross-species transmission and evolution of R-clamp categories.
The pie charts on the left illustrate the relative fraction of each R-clamp category in SIVcpz, SIVgor and HIV-1 groups M, N, O and P. Putative cross-species transmission events are indicated by colored arrows. The lower part of the figure shows a phylogenetic analysis of selected primate lentiviral Nefs is shown. Inferred most recent common ancestors are indicated by stars. In both images, R-clamp categories I, II, III, and IV are shown in blue, yellow, grey and orange, respectively.