[Abstract unavailable

The small (S), middle (M) and large (L) envelope proteins of the hepatitis B virus (HBV) are initially synthesized as multispanning membrane proteins of the endoplasmic reticulum membrane. We now demonstrate that all envelope proteins synthesized in transfected cells or in a cell-free system adopt more than one transmembrane orientation. The L protein disposes its N-terminal preS domain both to the cytoplasmic and the luminal side of the membrane. This unusual topology does not depend on interaction with the viral nucleocapsid, but is preserved in secreted empty envelope particles. Pulse-chase analysis suggests a novel process of post-translational translocation leading to the non-uniform topology. Analysis of L deletion mutants indicates that the block to co-translational translocation can be attributed to a specific sequence within preS, suggesting that translocation of L may be regulated. Additional topological heterogeneity is displayed in the S region of the envelope proteins and in the S protein itself, as assayed in a cell-free system. S proteins integrated into microsomal membranes exhibit both a luminal and a cytoplasmic orientation of the internal hydrophilic region carrying the major antigenic determinants. This may explain the unusual partial glycosylation of the HBV envelope proteins.

Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor ␥2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role ␥2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess ␥2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed ␥2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of ␥2-adaptin.In order to be released from cells, nonlytic enveloped viruses must undergo budding from either the plasma membrane or intracellular membranes, followed by pinching off or fission. The fission event is facilitated by virus-encoded late assembly domains that recruit and hijack the cellular budding machinery of multivesicular bodies (MVBs) (3, 31). In the cell, MVBs have the unique ability to generate intraluminal vesicles that bud away from the cytosol, a process topologically equivalent to that of enveloped virus budding. Sorting of cellular proteins toward internal MVB vesicles for either degradation, lysosomal functions, or exosomal release requires the coordinated actions of at least three hetero-oligomeric complexes, referred to as ESCRT (endosomal sorting complex required for transport) complexes I, II, and III (ESCRT-I, -II, and -III) (1,2,20,35,40). They are sequentially recruited to the late endosomal membrane and drive the formation of MVBs. After sorting, the process is terminated by the AAA-type ATPase Vps4, which disassembles and thereby recycles the ESCRT machinery. The functional loss of individual subunits of this machinery results in a malformed, dysfunctional MVB known as the "class E compartment" (4, 9, 11).In recent years, intense research has begun to uncover how enveloped RNA viruses utilize components of th...

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.

Hepatitis B virus (HBV) is a major cause of liver disease. Due to the tiny size of its genome, HBV depends on the critical interplay between viral and host factors for the generation of new viral particles from infected cells. Recent work has illuminated a multiplicity of spatially and temporally coordinated virus-host interactions that accompany HBV particle genesis. These interactions include the requirement of cellular chaperones for the maturation of the three viral envelope proteins, the cellular factors involved in dynamic modification, maturation, and intracellular trafficking of the nucleocapsids, and the host components of the multivesicular body (MVB) pathway enabling virion budding at intracellular compartments. Beside infectious virions, HBV produces at least two other types of particles, subviral empty envelope particles and subviral naked capsid particles, likely as a result of the engagement of different host factors by the viral structural proteins. Accordingly, HBV exploits distinct cellular pathways to release its particle types. Here, I review recent progress in these areas of the cell biology of HBV genesis.