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.
For functional diversity, the large (L) envelope protein of hepatitis B virus (HBV) acquires a dual transmembrane topology via co-translational membrane integration of the S region and partial post-translational translocation of the preS subdomain. Because each process requires the second transmembrane segment (TM2), we explored the action of this determinant by using protease protection analysis of mutant L proteins. We demonstrated that neither the disruption of a leucine zipper-like motif by multiple alanine substitutions nor the flanking charges of TM2 affected the topological reorientation of L. The dispensability of both putative subunit interaction modules argues against a link between preS post-translocation and envelope assembly. Phenotypic mixing experiments revealed that the preS and S protein domains of the related duck HBV L polypeptide failed to substitute functionally for the topogenic elements of HBV in directing the correct L topogenesis, implicating different translocation mechanisms used by the two hepadnavirus genera.Owing to the small size of their genome, viruses have evolved by retaining a maximum amount of information in a minimum of polypeptide sequences. As a consequence, many of the proteins or protein domains encoded by viruses are multifunctional. The large (L) envelope protein of hepatitis B virus (HBV), the prototype member of the Hepadnaviridae family, is an example of such multifunctionality. This protein serves in virus entry as a receptor protein and in virus assembly as a matrix-like protein and performs regulatory functions in addition (reviewed by Bruss et al., 1996). The multifunctionality depends on a dual membrane topology, a phenomenon first observed for the hepadnaviral L glycoprotein (Bruss et al., 1994;Ostapchuk et al., 1994;Prange & Streeck, 1995;Guo & Pugh, 1997;Swameye & Schaller, 1997). More recently, however, there is accumulating evidence that other viral coat proteins, such as the membrane protein of transmissible gastroenteritis coronavirus (Escors et al., 2001), subdomains of the envelope E1 and E2 proteins of hepatitis C virus (Cocquerel et al., 2002) and the fusion protein of Newcastle disease virus (McGinnes et al., 2003), also exist in two or more topological isoforms with separate functions.At present, little is known about the mechanism by which the dual topology of the HBV L protein is established. During biogenesis, the L protein, together with the related middle (M) and small (S) envelope proteins, are expressed from a single open reading frame by differential translation initiation. As a result, the sequence of S is repeated at the C termini of M and L, which contain the additional preS2 domain or preS2 and preS1 domains, respectively (reviewed by Heermann & Gerlich, 1991). All three proteins are cotranslationally integrated into the endoplasmic reticulum (ER) membrane, most likely directed by a signal-anchor and stop-transfer sequence encoded within the first and second transmembrane (TM) segments (TM1 and TM2) of their S domains (Eble et al., 1987...
Fragile DNA were found in a population of sperm associated with poor capacitation characteristics and apoptosis was observed after heat treatment. The results suggested that sperm dysfunction might be due to apoptotic sperm DNA resulting from an elevated temperature in the surroundings. The data suggested that the second population of high capacitating sperm induced chaperones such as heat shock proteins hsp 70 to protect against apoptosis.
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