Abstract
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.
Highlights
Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization
The asymmetric unit consists of two Nef/Hck heterodimers that assemble at the Nef–SH3 domain interface with the C-terminal flexible loop of Nef exposed in opposite directions (Supplementary Fig. 1)
In this study, we analyzed how lentiviral Nef proteins interact with dileucine-based sorting motifs to promote internalization of the T cell receptor (TCR) CD4 and CD3 from the surface of infected cells
Summary
Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. CD4 and the TCR–CD3 complex are constantly internalized via clathrin-mediated endocytosis, in the absence of infection This involves the interaction between sorting motifs in the cytoplasmic tails of these receptors and the assembly polypeptide 2 (AP2) complex[19, 20]. Nef stimulates clathrin-mediated endocytosis of CD3, CD4 and other cellular receptors by acting as an adapter of the adapter protein machinery[26] It contains an N-terminal membrane anchor domain of 60–120 amino acids length followed by a core domain of 130–150 amino acids, containing a highly conserved dileucine-based sorting motif at the center of a C-terminal flexible. 0.009 1.40 Most favored: 96.28 Allowed: 3.72 5NUI aValues in parentheses correspond to the highest resolution shell (2.55–2.50 Å or 2.83–2.78 Å, respectively) loop[27] This exposed sorting motif enables Nef to interact with AP2 and to induce the internalization of its target receptors. The structural basis for the fundamental differences in the ability of primate lentiviral Nefs to downmodulate CD3 is poorly understood
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