The envelope glycoprotein (Env) of human immunodeficiency virus, type 1 (HIV-1) undergoes rapid internalization after its transport to the cell surface. Env internalization is dependent upon information contained within the cytosolic domain of the protein. Here, we report that the cytosolic domain of Env binds specifically to the medium chain, 2, of the clathrin-associated protein complex AP-2, as well as to the complete AP-2 complex. 1 plays a critical role during the viral life cycle by mediating the attachment of virions to target cells and the fusion of viral and cellular membranes (1). Incorporation of Env, therefore, is essential for the formation of infectious viral particles. The cytosolic domain of the Env transmembrane subunit gp41 is the portion of the Env protein complex that is most likely to interact with the internal structural proteins of the virus (2-5). Although the cytosolic domain of Env is absolutely required for viral dissemination in vivo it seems to be dispensable for envelope incorporation into virions and, consequently, for viral replication in vitro (6). How newly assembled virions specifically acquire Env remains therefore largely unknown.An intriguing characteristic of the Env proteins of HIV-1 and simian immunodeficiency virus is that they undergo rapid endocytosis after their transport to the cell surface (7,8). As a consequence, the internal structural proteins of these viruses need to compete with the internalization machinery of the cell in order to acquire Env (9).2 Although the functional significance of this phenomenon is not understood, it is clear that Env behaves like other plasma membrane proteins that are rapidly internalized from the cell surface. Rapid internalization involves recruitment of plasma membrane proteins to clathrincoated pits, a process that is mediated by interaction of endocytic signals found in the cytosolic domains of the proteins with the clathrin-associated adaptor complex AP-2 (10 -12). The AP-2 complex consists of two large chains (␣ and ␤2), a medium chain (2), and a small chain (2). A direct interaction between 2 and tyrosine-based sorting signals from the cytosolic domains of several cellular integral membrane proteins has been recently demonstrated (13)(14)(15).To assess the diverse functions of the cytosolic domain of Env including its role in internalization from the plasma membrane, we are analyzing its interaction with cellular and viral proteins. Anti-Env antibodies allowed us to co-immunoprecipitate Env with the AP-2 complex from HIV-1-infected lymphocytes, demonstrating that these proteins associate in vivo. Using GST-Env tail fusion proteins, we then identified the 2 chain of AP-2 as a protein that interacts with the cytosolic domain of Env. Binding of 2 to the cytosolic domain of Env was dependent on the presence and the context of a tyrosinebased sorting motif that is crucial for Env internalization (7), but it was also influenced by a glycine residue that had not previously been identified to be important for efficient endosomal sort...

The envelope glycoprotein (Env) is an essential component of retroviruses because it mediates the selective attachment of virus to its target cell (19). Env is not necessary for the formation and the release of retroviral particles, but Env of lentiviruses, like the glycoproteins of other enveloped RNA viruses, has been implicated in the spatial restriction of virus production (2, 5, 51). In addition, Env contributes to controlling the rate with which virions exit the host cell (44, 50). Env's position in the viral replication cycle is thus pivotal not only because it controls viral entry but also because it regulates when and where exactly virus will be released during the late phase of the viral life cycle.Polarization of lentiviral release is observed not only in epithelial cells but also in lymphocytes and probably in neurons (8,24,41,55). Such a directed release of virus may be particularly important for an efficient spread of virus in the crowded environment of lymph nodes, where infection is propagated locally, but polarized secretion is probably also important during systemic dissemination, when the virus crosses tissue barriers.How Env targets virus release to certain areas of the cell surface is not known. However, viral exit at distinct sites coincides with Env localization in these areas. It seems likely, therefore, that Env accumulation at distinct sites is a prerequisite for the polarized release of infectious particles. Consequently, signals which direct Env from the trans-Golgi network (TGN) to these sites are crucial for directional virus secretion (see, e.g., references 8 and 31).To elucidate how Env targets virus release to particular areas of the cell surface, we analyzed its trafficking during the late phase of the viral replication cycle. As part of these investigations we characterized the interaction of Env's cytosolic domain (EnvCD) with proteins of the cellular sorting machinery. The targeting of membrane proteins from the Golgi apparatus to the plasma membrane or to compartments of the endosomal system is mediated by specialized clathrin-coated vesicles (CCVs) (22,26,32,35,45). Endocytosis of membrane proteins gives rise to the formation of similar transport vesicles at the plasma membrane. Selection of the membrane proteins into CCVs largely depends on the interaction of signals in the cytosolic domains of these proteins with specific adapter proteins (APs). These APs are part of the vesicle coats forming at the cytosolic leaflet of the lipid bilayer. The existence of four

The envelope glycoprotein (Env) of HIV-1 interacts with the clathrin-associated adaptor complex AP-2 during the late phase of the viral replication cycle. Upon its synthesis, Env, therefore, is retrieved from the cellular surface unless internalization is inhibited by viral Gag. Here we demonstrate that not only Env, but also HIV-1 Gag, specifically binds to AP-2. Gag-AP-2 association was found to depend on tyrosine residue 132 and valine residue 135 at the matrix-capsid junction in the Gag polyprotein. Results of a morphological analysis of viral egress from cells expressing dominant-negative AP-2 suggest an involvement of AP-2 in confining HIV-1 exit to distinct microdomains. Further, particle release from AP-2-mutant cells was enhanced compared to release from wild-type cells but the infectivity of virus released from these cells was moderately reduced. Together these data attribute a role to the AP-2 complex in the regulation of HIV-1 assembly/release.

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adapter proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adapter complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN-and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.

Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adapter proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adapter complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN-and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.