Clathrin-Mediated Post-Golgi Membrane Trafficking in the Morphogenesis of Hepatitis Delta Virus

Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.Pathogens often take advantage of intracellular pathways involved in the trafficking of cellular macromolecules in order to carry out their life cycle, which consists of virus entry, translation, genome replication, assembly, and release. The clathrin-mediated endocytic route is a pathway commonly used for virus entry (29). Following clathrin-mediated endocytosis, incoming viruses are transported together with their receptors from the plasma membrane into early and late endosomes. Several links between clathrin adaptor complexes and viral biogenesis, including those of influenza virus (37), reovirus (13), and vesicular stomatitis virus (33), have been demonstrated.Clathrin and its adaptor proteins (APs), which constitute the major components of clathrin-coated vesicles (CCVs), are often the carriers of proteins and lipids that are transported from the trans-Golgi network (TGN) to the endosome (20, 35). Clathrin-mediated exocytosis has been found to participate in viral multiplication. The envelope protein of vesicular stomatitis virus, glycoprotein 1, recruits clathrin adaptor complex adaptor protein 1 (AP1) onto Golgi membranes and possibly leaves the TGN in CCVs for subsequent transport to endosomes (1). It is also known that interaction of AP1 with the matrix domain of human immunodeficiency virus type 1 Gag protein promotes viral release (5). In addition, Vpu inhibits the endosomal accumulation of the human immunodeficiency virus type 1 structural proteins Env and Gag, which is known to enhance viral assembly and release at the plasma membrane (39). Furthermore, large hepatitis delta antigen (HDAg-L) encoded by the hepatitis delta virus (HDV) has recently been identified as a novel clathrin adaptor-like protein (18). HDAg-L specifically interacts with clathrin heavy chain (CHC) at the TGN and inhibits clathrin-mediated protein transport. However, the role of CHC in the life cycle of HDV remains unclear.HDV is a highly pathogenic virus. The virion is coated with the envelope proteins of hepatitis B virus (HBV), the hepatitis B virus surface antigens (HBsAgs) (24). Superinfection or coinfection with HBV may result in fulminant hepatitis and progressive chronic liver cirrhosis (3, 36). The small HDAg (HDAg-S) lacks the unique C-terminal 19-amino-acid sequence of HDAg-L (6, 41, 43) and functions as a transactivator of HDV genome replication in the nucleus (23, 24). Both HDAg-S and HDAg-L possess nuclear localization signals (NLSs) spanning amino acid residues 35 to 88 and are mainly localized in the nuclei of transfected cells in the absence of HBsAg (7, 8). However, HDAg-L has been demonstrated to be a nucleocytoplasmic shuttling protein with a nuclear export signal (NES) at its unique C terminus, and this is important for HDV assembly (27). In the presence of HBsAg, HDAg-L relocalizes to the cytoplasm (29). In addition, a NES-interacting protein of HDAg-L, NESI, has been identified to be essential for the HDAg-L-mediated nuclear export of HDV RNA (42). Furthermore, the proline-rich motif within the unique 19-amino-acid extension together with isoprenylation of the CXXX motif (15) are essential for HDAg-L to form delta virus-like particles (VLPs) with HBsAg (19, 22). Taken together, these results imply that an intracellular association between HDAg-L and HBsAg in the cytoplasm is the driving force of HDV assembly. The interaction of HDAg-L with HBsAg facilitates the assembly and secretion of HDV particles. Nevertheless, the cellular proteins and pathways involved in the transport, packaging, and secretion of HDV are poorly understood.In this study, the involvement of clathrin-mediated trafficking in the propagation of HDV is biochemically characterized. Downregulation of functional CHC significantly reduced the efficiency of the CCV-mediated HDV assembly. However, CHC is not essential for the assembly of HBV subviral particles (SVPs). These results indicate that, although HBV and HDV share common surface antigens, different mechanisms are involved in their viral assembly and release. In addition, the assembly efficiency of the various HDV genotypes correlates well with the ability of HDAg-L to interact with CHC. This may reflect the fact that there is lower pathogenicity among patients infected with HDV genotype II than among those infected with genotype I.