Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3

Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.

Binding of Hck SH3-E to SIV mac239 Nef and complex assembly. (a) Interactions between the Hck SH3-E domain (green) and SIV mac239 Nef (white/blue). The RT loop sequence of the SH3 domain has been engineered for high affinity binding to Nef and was modified to E 90 GWWG 31 . The two tryptophanes mediate interactions with residues on helix 4 of Nef. The key interactions of the P 104 xVPxR motif of SIV Nef are displayed. The salt bridge formation of R109 Nef to D96 Hck is a major determinant for binding. Important interacting residues are shown in stick representation. (b) Assembly of the Nef-Hck heterodimer in the asymmetric unit cell of the crystal structure. Of note, the crystallographic dimer formed by R105 and D123 of Nef NL4-3 , corresponding to the conserved residues R137 and D155 in SIV mac239 Nef is not observed in this structure.

SUPPLEMENTARY INFORMATION
Supplementary Figure 2 Sequence alignment of SIV mac239 Nef and HIV-1 Nef SF2 and secondary structure display. The sequences of SIV mac239 Nef and HIV-1 Nef SF2 share 34.5% identity and 52.9% similarity. Insertions in the SIV allele relative to the HIV-1 sequence are located around position 30, a highly charged stretch of 19 residues (3 basic and 8 acidic residues) from amino acids 79 to 97 preceding the PxxP motif, and 25 residues at the very C-terminus. The three key elements of Nef proteins, the myristoylation motif MGxxxS at the N-terminus, the central PxPxR motif, and the dileucine-based sorting motif ExxxL in the C-terminal flexible loop, are conserved and highlighted in red. Charged residues flanking the ExxxL sorting motif in the C-terminal flexible loop are boxed. The secondary structures of SIV mac239 Nef determined here (PDB accession code 5NUI) and of HIV-1 Nef SF2 (assembled from protein structures 1QA5, 3REA, and 3RBB) are displayed above and below the sequences, respectively.

Supplementary Figure 3
Size exclusion chromatography of the tripartite complex between SIV mac239 Nef, an SH3 domain, and the cytoplasmic tail of CD3 . The SIV mac239 Nef (66-235) protein, termed SIV-B, eluted as a homogeneous peak at its apparent size of 23.4 kDa. Addition of the SH3 domain from Hck, engineered in the RT loop for optimized binding to SIV Nef, termed SH3 E , only slightly increased the size of the complex, similarly as observed before 33 . Addition of CD3  (71-135) containing the two SIV Nef interaction domains (SNID1 and SNID2) led to complex formation with Nef. Likewise, addition of CD3  to the preformed SIV Nef-SH3 E domain complex led to formation of the tripartite complex. The analytical gel filtration was performed using a Superdex S75 (10/300 GL) column (GE Healthcare) on a multicomponent Waters 626 LC system (Waters, MA). Elution profiles were run in 20 mM HEPES (pH 8.0), 100 mM NaCl, and 1 mM TCEP at room temperature.

Supplementary Figure 4
Orientation of the sorting motif helices relative to SIV Nef. The structures of SIV mac239 Nef bound to the CD3  complex (PDB accession code 3IK5) 35 or bound to the dileucine-based sorting motif ExxxLM are shown as cartoon model in orange/red and light blue/blue, respectively. The structures were aligned for the Nef core domain (inset). The N-to C-terminal direction of the bound helices of the YxELxL motif (red) and the ExxxLM motif (blue) is similar but the orientation of the helix relative to Nef varies by 48°.

Supplementary Figure 5
Importance of the di-arginine motif for Nef-mediated downmodulation of CD4, CD3, and MHC-I. Jurkat T cells were transfected with bicistronic vectors coexpressing the indicated nef alleles and GFP, and assayed for surface expression of CD4, CD3, and MHC-I by flow cytometry. Receptor surface expression was determined in cells expressing no, low, medium (med), or high levels of GFP as described in the Methods section. The results of one representative experiment are shown.

Supplementary Figure 6 Nef expression in transfected HEK293T cells and infected
PBMCs. (a) HEK293T cells were transfected with expression vectors for the indicated Nef proteins. Two days post transfection cells were lysed and analyzed by Western blotting. Nef was detected using a rabbit antiserum directed against HIV-1 Nef. GAPD1 served as loading control. (b) PBMCs were infected with HIV-1 SF2 constructs expressing the indicated Nef proteins. Three days post infection, cells were analyzed by Western blotting as described for (a). HIV-1 Gag was detected using an antiserum against HIV-1 p24. (c) Nef expression levels in transfected HEK293T cells (n=1) and infected PBMCs (n=2) were quantified and normalized to GAPDH and HIV-1 Gag, respectively. Pearson's correlation coefficient was calculated.

Supplementary Figure 7
HIV-1 Nef gain-of-function mutations for CD3  binding. Based on the sequence of HIV-1 Nef SF2 of 210 amino acids, single point mutations and sequence exchanges were generated for the acquisition of CD3 down-regulation, using the sequence of SIV mac239 Nef as template for gain-of-function. First, point mutations were introduced in the hydrophobic crevice of Nef ranging from residues 80 to 130. Next, the Nterminal anchor domain and C-terminal flexible loop section were exchanged. These sequence stretches were further optimized to determine the minimally required regions. Finally, the YxxL and ExxxLL motifs were mutated to alanines to unravel the functions of these sorting signals for Nef mediated endocytosis.

Supplementary Figure 8 ITC measurements between Nef gain-of-function variants D
and P and CD3  ITAM motifs. (a) Nef-D and (b) Nef-P proteins were expressed as recombinant proteins with domain boundaries 23-210, C210A. Both proteins contain the 18 mutations in the 4/5 core domain of Nef. Nef-P contains in addition the N-terminal YxxL motif. The affinity of Nef-P for the second ITAM motif of CD3  is only about 2-fold higher than that of Nef-D. The significantly increased ability to down-regulate CD3 by Nef-P compared to Nef-D is therefore not a result of much better binding to CD3, but rather supposed to be due to the improved interaction with the adaptor protein machinery.