Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular ␥2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered ␥2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same ␥2-adaptor and that disruption of the HBV/␥2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of ␥2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both ␥2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with ␥2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.

␥2-Adaptin is a putative member of the clathrin adaptor protein family with unknown physiological function. We previously reported that ␥2-adaptin acts as a ubiquitin receptor by virtue of its ubiquitin-interacting motif. Here we demonstrate that this motif mediates a specific physical interaction with the ubiquitin ligase Nedd4 and promotes ubiquitination of ␥2-adaptin. By mapping regions of Nedd4 involved in binding to ␥2-adaptin, we identified its C2 domain to be essential, whereas the WW and HECT domains are dispensable. Consistent with this, we uncovered that the C2 domain of Nedd4 is ubiquitinated itself and as such is recruited by the ubiquitin-interacting motif of ␥2-adaptin for subsequent ubiquitin conjugation. Unlike known coupled ubiquitination reactions, this novel type of interaction leads to mono-and multi/polyubiquitinated ␥2-adaptin. In addition, we show that ␥2-adaptin functions in the endosomal/ multivesicular body (MVB) pathway. Depletion of ␥2-adaptin impairs the degradation of internalized epidermal growth factor and results in defective MVB morphology characterized by significantly enlarged vesicles. These defects cannot be rescued by ␥1-adaptin, a closely related homolog of ␥2-adaptin, which is unable to bind ubiquitin. Together, these results indicate that ␥2-adaptin may operate within the MVB sorting system in a manner different from that of classic adaptins.The covalent attachment of ubiquitin marks proteins for various cellular fates and functions, including proteasomal degradation, endocytosis, endosomal sorting, DNA repair, and virus budding. One way cells interpret and transmit the information conferred by ubiquitin is through proteins that bind ubiquitin noncovalently. These ubiquitin receptors contain one or more ubiquitin binding domains (UBD), 2 of which at least sixteen have been identified to date (1, 2). The first discovered UBD was the ubiquitin-interacting motif (UIM) that is found in several ubiquitin receptors controlling endocytic membrane traffic (3). This class of proteins, including eps15, epsin, Hrs, and Stam, regulates the internalization of plasma membrane proteins into the endocytic pathway as well as the sorting of proteins into the multivesicular body (MVB) in a ubiquitin-dependent manner (4 -8). A recent addition to this group of UIM-containing receptors is ␥2-adaptin, which we originally identified as a hepatitis B virus (HBV) interacting protein in a yeast two-hybrid screen (9, 10).␥2-Adaptin is classified as a member of the heterotetrameric clathrin adaptor complex (AP) family (11). APs mediate the sorting of protein cargo and their incorporation into clathrincoated transport vesicles. Four AP complexes have been identified designated AP-1 through AP-4, and they all exhibit a similar organization consisting of two large subunits, a medium-sized subunit, and a small subunit (11, 12). ␥2-Adaptin is highly similar to ␥1-adaptin (one large subunit of the transGolgi network/early endosome adaptor AP-1) in both primary sequence and modular domain architecture (13,...