A DILEUCINE MOTIF IN HIV-1 NEF ACTS AS AN INTERNALIZATION SIGNAL FOR CD4 DOWN-REGULATION AND BINDS THE AP-1 CLATHRIN ADAPTER

Human immunodeficiency virus 1 (HIV-1) Nef downregulates surface expre ssion of CD4, an integral component of the functional HIV receptor com plex, through accelerated endocytosis of surface receptors and diminis hed transport of CD4 from the Golgi network to the plasma membrane [1- 3]. HIV-1 Nef also diminishes surface expression of major histocompati bility complex (MHC) class I antigens [4]. In the case of HIV-2 and si mian immunodeficiency Virus 1 (SIV-I) Nef, aminoterminal tyrosine-base d 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 o f the endocytic machinery is unknown. Here, we identify a highly conse rved 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 Il-amino-acid region from Nef containing the dileucine motif. Fina lly, HIV-1 Nef binds to the AP-1 adaptor, both in vitro and in vivo, i n a dileucine-dependent manner. We conclude that this conserved dileuc ine motif in HIV-1 Nef serves as a key interface for interaction with components of the host protein trafficking machinery. Our findings als o reveal an evolutionary difference between HIV-1 and HIV-2/SIV in whi ch the Nef proteins utilize structurally distinct motifs for binding c ellular adaptors.