γ2-Adaptin, a Novel Ubiquitin-interacting Adaptor, and Nedd4 Ubiquitin Ligase Control Hepatitis B Virus Maturation

Abstract

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.

Involvement of host cellular multivesicular body functions in hepatitis B virus budding

“…The p9 YPDL domain that binds AIP1 also binds the clathrinassociated AP2 adaptor complex that sorts cargo proteins into coated pits for endocytosis, and this interaction is important for EIAV budding (55). Interestingly, HBV envelope proteins S and L were recently reported to interact with, respectively, AP2 (10) and ␥2-adaptin, a protein related to the large subunits of vesicle adaptor complexes such as AP2 (9,56). Although the possible function of S-AP2 interaction is not known, L-␥2-adaptin interaction appears important for HBV virion production (56).…”

“…Interestingly, HBV envelope proteins S and L were recently reported to interact with, respectively, AP2 (10) and ␥2-adaptin, a protein related to the large subunits of vesicle adaptor complexes such as AP2 (9,56). Although the possible function of S-AP2 interaction is not known, L-␥2-adaptin interaction appears important for HBV virion production (56). Thus, like EIAV, HBV budding may use host functions not only in late endocytic/MVB pathways but also in early endocytic pathways.…”

Assembly and budding of a hepatitis B virus is mediated by a novel type of intracellular vesicles

“…These findings support and extend the data of a recent work which showed that human HBV budding involves an endosomal compartment. 18 Why do some viruses bud into endosome-like vesicles? The endosomal system clearly plays a significant role in the assembly of many retroviruses, 24 preformed HIV particles from late endosomes are infectious.…”

“…Recently, some chaperones and ␥2-adaptin were identified as host factors participating in HBV progeny formation. [16][17][18] Viruses exploit a broad arsenal of strategies to assemble and bud within cells. For example, Pox-like, Irido-like, and Asfar-like viruses induce the formation of "aggresomes," which serve as scaffolds to concentrate structural components.…”

Estudo da função de AP1y2 e Alix no direcionamento de proteínas para degradação em lisossomos ou liberação em vesículas extracelulares

“…The siRNA were purchased from Dharmacon (Lafayette, CO) as nucleotide duplexes with 3′dTdT overhangs, designed to target human γ1 (5′-GGAAGAGCCUAUUCAGGUA-3′), γ2 (5′-AAACCCUGCUUUGC-UGUUAA-3′) (Rost et al, 2006) and μ1A (5′-GGCAUCAAGUAUCGG-AAGA-3′) (Janvier and Bonifacino, 2005). An alternative siRNA targeting γ2 (γ2 siRNA#2; 5′-CACAGAACACAGCAUAUCU-3′), with a 3′dTdT overhang, was purchased from Bioneer Inc (Alameda, CA).…”