A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor

Abstract

Human immunodeficiency virus 1 (HIV-1) Nef downregulates surface expression of CD4, an integral component of the functional HIV receptor complex, through accelerated endocytosis of surface receptors and diminished transport of CD4 from the Golgi network to the plasma membrane [1-3]. HIV-1 Nef also diminishes surface expression of major histocompatibility complex (MHC) class I antigens [4]. In the case of HIV-2 and simian immunodeficiency virus 1 (SIV-1) Nef, aminoterminal tyrosine-based motifs mediate the binding of Nef to the AP-1 and AP-2 adaptors and this interaction appears to be required for CD4 downregulation [5,6]. As these tyrosine motifs are not present in the HIV-1 Nef protein, the molecular basis for the presumed interaction of Nef with components of the endocytic machinery is unknown.Here, we identify a highly conserved dileucine motif in HIV-1 Nef that is required for downregulation of CD4. This motif acts as an internalization signal in the context of a CD8-Nef chimera or in a fusion of the interleukin-2 receptor a with an 11-amino-acid region from Nef containing the dileucine motif. Finally, HIV-1 Nef binds to the AP-1 adaptor, both in vitro and in vivo, in a dileucine-dependent manner. We conclude that this conserved dileucine motif in HIV-1 Nef serves as a key interface for interaction with components of the host protein trafficking machinery. Our findings also reveal an evolutionary difference between HIV-1 and HIV-2/SIV in which the Nef proteins utilize structurally distinct motifs for binding cellular adaptors.

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

“…The expression plasmid IL2R-LL encodes the outer and transmembrane region of the IL-2 receptor linked to the di-leucine-based motif from Nef as cytoplasmatic tail (Bresnahan et al, 1998). Tac-DKQTLL (Letourneur and Klausner, 1992) and pTTMb (Marks et al, 1996) contain the di-leucine-based motifs from CD3␥ and the tyrosine-based motif from HLA-DM␤, respectively.…”

“…To this end, we determined if the expression of our fragments could interfere with the internalization of the extracellular and transmembrane portions of the IL2 receptor linked to the flexible loop of the HIV-1 Nef protein and therefore containing a cytoplasmic dileucine-based motif (IL2R-LL; Bresnahan et al, 1998) and determined its subcellular localization. NIH3T3 cells were cotransfected with plasmids encoding for the IL2R-LL fusion protein and the Myc-tagged di-leucine binding fragments, respectively.…”

“…Several proteins have been proposed to direct the sorting of Nef. They include the ␤-chain of AP-1 complexes (Bresnahan et al, 1998;Greenberg et al, 1998), the ␤-subunit of the COP-I coatomer (Piguet et al, 1999;Janvier et al, 2001), and the regulatory subunit H (V1H) of the vacuolar ATPase (VATPase), previously named NBP1 for Nef binding protein 1 (Lu et al, 1998;Mandic et al, 2001). Although the precise sites of these interactions varied, all protein complexes were reported to bind to the C-terminal flexible loop in Nef, which contains the di-leucine-based motif at its center (Grzesiek et al, 1996;Lee et al, 1996;).…”

Identification of a Motif in the Carboxyl Terminus of CXCR2 That Is Involved in Adaptin 2 Binding and Receptor Internalization

“…A highly conserved carboxyl-terminal Leu-Leu motif has been demonstrated to involve the endocytosis of various membrane proteins (20)(21)(22), including G protein-coupled β 2 …”

Protéines Nef du VIH et K3/K5 du virus associé au sarcoma de Kaposi : des « parasites »de la voie d’endocytose

“…Les travaux réalisés au cours de ces dernières années ont permis des avancées significatives pour la compréhension des mécanismes responsables des perturbations du trafic intracellulaire induites par Nef. En effet, Nef interagit directement avec les sous-unités µ et β des complexes AP-1, AP-2 et AP-3 [6,9,10], ainsi qu'avec la sous-unité β des complexes COPI (coatomer protein complex I) [11,12] impliqués dans le trafic vésiculaire au sein de la voie de sécrétion, mais qui interviennent également dans le transport entre endosomes précoces et tardifs [13]. Des interactions de Nef avec la sous-unité catalytique de l'ATPase vacuolaire (V-ATPase) nécessaire à l'acidification des compartiments endosomiques, ainsi qu'avec la protéine PACS-1 ont également été rapportées [14,15].…”