The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation

Abstract

The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.

Modulation of Cellular Protein Trafficking by Human Immunodeficiency Virus Type 1 Nef: Role of the Acidic Residue in the ExxxLL Motif

“…Notably, the concept that the sorting motifs in target proteins may interact directly with AP complexes during Nef-mediated modulation has been supported by two recent studies, each of which reported that the LL sequence in the cytoplasmic domain of CD4, while required for Nef-mediated down-regulation, does not contribute to the interaction between Nef and CD4 (2,6). In this emerging model, the AP binding motifs in both Nef and the target protein are functional.…”

Human Immunodeficiency Virus Type 1 Nef: Adapting to Intracellular Trafficking Pathways

“…A complex of full-length Nef with the CD4 cytoplasmic tail was found to be more stable, with a dissociation complex in the submicromolar range, and suggested that other amino acids in the N terminus contributed (126). In vivo there may be other factors that further stabilize and alter this interaction, because the dileucine motif was not needed when the interaction was detected in vivo (16,30).…”

“…Indeed, Nef has been shown to bind to the CD4 cytoplasmic tail in yeast two-hybrid assay systems (133), in purified protein assay systems (68,71,126), in cell lysates (80), and in living cells (16,30). An NMR structural analysis demonstrated that the direct physical interaction occurred between a hydrophobic pocket in the Nef core domain and a 13-amino-acid peptide (QIKRLL SEKKT) from the CD4 cytoplasmic tail (68).…”

Nef-Induced CD4 Endocytosis in Human Immunodeficiency Virus Type 1 Host Cells: Role of p56lckKinase

“…Nef-induced CD4 downregulation is known to be independent of Ser phosphorylation (20) and is therefore governed by mechanisms different from those involved in PMAinduced CD4 downregulation. However, the Leu-based sorting motif in the CD4 cytoplasmic tail is critical for both PMA and Nef-induced CD4 downregulation (2,5,24,31,56,60,68), thus indicating that despite being different, the mechanisms involved in Nef-and PMA-induced CD4 downregulation partially overlap.…”