In vitro analyses of Nef-AP-2 interaction determinants. (A) Purified recombinant proteins used in this experiment include hexahistidine-tagged wild-type and DD174,175AA mutant Nef, AP-2 core tagged with GST at the C terminus of α and hexahistidine at the N terminus of β2, and the ear domain of AP4 ɛ tagged at its N terminus with GST. Binding of Nef proteins to GST-tagged AP-2 core and ɛ-ear was analyzed by GST pulldown and SDS-PAGE, followed by Coomassie blue staining (top) or immunoblotting (IB) with an antibody to Nef (bottom). The input recombinant proteins were run alongside for comparison (first four lanes). Wild-type hexahistidine-tagged Nef is visible as a ∼27-kDa band in the fifth lane. This experiment is representative of three experiments with similar results. (B) GST-ɛ-ear (control) or wild-type or DD174,175AA mutant hexahistidine-tagged Nef was covalently attached to the surface of a CM5 sensor chip. Subsequently, the surfaces were probed for binding of untagged AP-2 core by SPR. Shown are control-subtracted sensorgrams of individual injections of AP-2 core (25 μM) over surfaces containing wild-type or DD174,175AA mutant hexahistidine-tagged Nef. (Inset) Plot of varying concentrations (0.5 μM to 25 μM) of AP-2 core. (C) The interaction of hexahistidine-tagged Nef with GST-tagged AP-2 core is sensitive to ionic strength. GST-tagged AP-2 core was incubated with hexahistidine-tagged Nef in the presence of increasing NaCl concentrations (0.1 M to 1 M) during the pulldown experiment. The positions of the recombinant proteins (for simplicity, tags are not indicated) are shown on the left and those of molecular mass markers (in kilodaltons) on the right of the gel.

Immunofluorescence microscopy analysis of CD4 downregulation by wild-type and mutant Nef proteins. HeLa cells were cotransfected with two different plasmids: pCMV.CD4 and wild-type or mutant versions of pCI.Nef. Cells were fixed, permeabilized, and stained with mouse anti-CD4, followed by Alexa Fluor 594-conjugated anti-mouse immunoglobulin G. The distribution of CD4 was imaged by confocal microscopy. Bar, 10 μm.

Y3H analysis reveals qualitative differences among dileucine motifs. (A) Sequences of the mouse tyrosinase cytosolic tail and the NL4-3 HIV-1 Nef flexible loop. The dileucine and diacidic motifs are underlined. Amino acid numbers are given to the left and right of the sequences. (B) Y3H analysis of the interaction of the α-σ2 hemicomplex with wild-type and mutant versions of the tyrosinase cytosolic tail and with mutant versions of full-length Nef. The Y3H experiments were performed as described in the legend to Fig. 1 and in Materials and Methods.

Identification of residues within the HIV-1 Nef C-terminal flexible loop that contribute to AP-2 binding. (A) Ribbon diagram and surface rendering of the 3-dimensional structure of HIV-1 Nef. Determinants for CD4 downregulation and AP-2 binding within the C-terminal flexible loop are highlighted in red. This model was generated with PyMOL (www.pymol.org) by using coordinates from accession codes 1QA5 (15) and 2NEF (21), with additional residues of the loop region modeled arbitrarily. The structure, sequences, and residue numbers correspond to the NL4-3 variant of HIV-1 Nef. (B) Plasmids used for Y3H experiments. Nef was expressed as a GAL4BD fusion protein from the pBridge vector along with the AP-2 σ2 subunit. AP-2 α (αC isoform) was expressed as a GAL4AD fusion protein from the pGADT7 vector. (C) Comparison of the loop sequence (residues 154 to 180) of NL4-3 Nef (GenBank accession number U26942) to an alignment of 1,290 HIV-1 and SIVcpz Nef sequences found on the Los Alamos HIV sequence database (www.hiv.lanl.gov). The NL4-3 residues were scored for the frequency of identity and similarity at each position within the alignment. Similarity was defined by a log odds score of ≥0 on the BLOSUM62 substitution matrix (24). The bar graph indicates the frequency of occurrence of each NL4-3 residue in the aligned sequences (red bars) as well as the frequency of similar residues at each position (pink bars). (D) Y3H analysis of binding of full-length NL4-3 HIV-1 Nef loop mutants to the AP-2 α-σ2 hemicomplex. Each colony represents an alanine substitution mutant at the corresponding residue of the Nef loop coexpressed with α and σ2 by cotransformation of HF7c yeast with the plasmids diagramed in panel B. The third position (residue 156) is a naturally occurring alanine in the wild-type sequence. Growth in the absence of histidine (−His) or in the combined absence of histidine and presence of 3 mM 3-aminotriazole (+3AT) is indicative of interactions at two levels of stringency. Immunoblot analysis showed that all the fusion proteins were expressed at similar levels in the transformed yeast cells (data not shown).

Mutational analysis of Nef binding to the AP-1 γ-σ1, AP-2 α-σ2, and AP-3 δ-σ3 hemicomplexes. Interactions were analyzed as described in the legend to Fig. 1 and in Materials and Methods.

FACS analysis of CD4 downregulation by wild-type and mutant Nef proteins. HeLa cells were cotransfected with pCMV.CD4 and either wild-type or mutant versions of pNL4-3 Nef.IRES.GFP, which is designed to express Nef and GFP as separate proteins from the same bicistronic transcript. For the FACS analyses, GFP fluorescence was used as a marker to identify cells that had been transfected with a Nef expression plasmid. (A) FACS histograms of CD4 surface levels in cells transfected with the plasmids mentioned above. For each condition, the relative amount of CD4 at the plasma membrane is shown for cells transfected with the pIRES2.GFP empty vector (no Nef expression) (thin lines) and for cells transfected with wild-type or mutant Nef (bold lines). The fluorescence profile of cells left untransfected (no CD4 expression) (solid gray shading) is also shown. (B) (Top) Immunoblot (IB) analysis of the expression of wild-type and mutant versions of Nef by using an antiserum to HIV-1 Nef. Cells transfected with the empty vector pIRES2.GFP (no Nef expression) were used as a control. (Bottom) Quantification of the results in panel A. Bars represent geometric means ± standard errors of the means from three independent experiments. Levels of CD4 on the surfaces of Nef-expressing cells are compared to those on the surfaces of cells transfected with the pIRES2.GFP empty vector (taken as 1). (C) FACS analysis of CD4 endocytosis in cells expressing wild-type or mutant versions of Nef. Data for cells that were transfected with the empty vector and do not express Nef are included as a control.