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.

Abstract. Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene produc0 contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg-and -ArgArg-). The fur-directed activity (fufin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or fufin with the mouse B-nerve growth factor precursor (pro-B-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo. p OSTTRANSLATIONAL proteolysis is a common mechanism required for the synthesis of biologically active proteins and peptides in all eukaryotes examined, including yeast (21), invertebrates (56), and mammalian cells (17,62). One of the early events in precursor protein maturation is endoproteolysis at the carboxyl side of pairs of basic amino acid sequences (especially -LysArg-and -ArgArg-). This type of endoproteolytic cleavage was initially inferred from the sequences of several endocrine and neuroendocrine precursor proteins and was first proposed from studies of proinsulin (9, 64) and the ACTH/B-endorphin precursor, proopiomelanocortin (POMC) t (11). Subsequent studies have revealed a broad spectrum of precursor proteins that require endoproteolysis at pairs of basic amino acids to yield mature peptides, including serum factors (3), viral polyproteins (43, 49, 51, 52), growth factors (24,26,58,75), and receptors (76).

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