Large Hepatitis Delta Antigen Is a Novel Clathrin Adaptor-Like Protein

Abstract

Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrinmediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin.Hepatitis delta virus (HDV) causes fulminant hepatitis and progressive chronic liver cirrhosis in patients superinfected or coinfected with hepatitis B virus (HBV) (1, 38). HDV particles are enveloped by the HBV surface antigen (HBsAg) (22), but replication of the viral genome is independent of the helper HBV (21). The HDV genome is a single-stranded circular RNA molecule of approximately 1.7 kilobases that encodes the only known HDV protein, hepatitis delta antigen (HDAg). Two forms of HDAg, small HDAg (HDAg-S) and large HDAg (HDAg-L), which contain 195 (24 kDa) and 214 (27 kDa) amino acid residues, respectively, were detected in the livers and sera of HDV-infected patients (5, 49). The HDAgs are translated from the same initiation codon of a single open reading frame (25) and share identical functional domains except for an additional 19-amino-acid motif at the C terminus of the HDAg-L resulting from RNA editing (33,34).HDAg-S is essential for the replication of HDV RNA (21), whereas HDAg-L is required for HDV assembly in a later stage of viral multiplication (4,8). The unique C-terminal domain of HDAg-L bears an isoprenylation motif (211-CRPQ-214). Isoprenylation and proline-rich motifs at the unique C terminus are important for the interaction of HDAg-L with HBsAg and the assembly of HDV (8,9,17,20). Both HDAg-S and HDAg-L possess nuclear localization signals (NLSs) spanning amino acid residues 35 to 88 and are mainly localized to the nucleus in the absence of HBsAg (6, 7). Nevertheless, in the presence of small HBsAg, HDAg-L localizes to both the nucleus and the cytoplasm. A nuclear export signal (NES) at the unique C terminus of HDAg-L was identified (24). Recently, we also identified a cellular protein, NESI, which specifically interacts with the NES of HDAg-L and is essential for HDAg-L-mediated nuclear export of HDV RNA (47...

Characterization of Hepatitis Delta Virus in Sub-Saharan Africa

“…Indeed, all our clade-1 strains harbored proline at position 205, while in our strains of the other clades, arginine was found in this position. Similarly, the clathrin box, thought to be important for virion packaging (31,32), varied between clade 1 (LFPSD) and our strains of the other clades (LPLLE). Furthermore, the isoprenylation signal, which is important for virion maturation (31), was CRPQ in clade 1 and CTPQ in our non-HDV-1 strains.…”

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

“…Plasmid pGEX-HDAg-L(198-210) has been described previously (18). For construction of plasmid pGEX-HDAg-L(198-210)-II, a cDNA fragment encompassing the C terminus of the genotype II HDAg-L from amino acid residues 198 to 210 was generated by annealing two synthetic oligonucleotides representing the two strands of HDAg-L(198-210) with additional recognition sequences of EcoRI/ SalI at the ends.…”

“…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.…”

Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen

“…Expression of GST-tagged fusion proteins in Escherichia coli BL21 and partial purification of His-tagged fusion proteins have been described previously (6). To perform a GST pulldown assay, GST fusion proteins precoupled to glutathione-Sepharose 4B beads (GE Healthcare Bio-Sciences) were incubated at 4°C overnight with partially purified His-tagged fusion proteins or total protein lysates prepared from cultured cells treated with PBST.…”

“…HDAg has two isoforms, small HDAg (HDAg-S; 195 amino acid [aa] residues) and large HDAg (HDAg-L; 214 amino acid residues). HDAg-S functions as a trans-activator in HDV replication (2,3), whereas HDAg-L contains a 19-aminoacid C-terminal extension which is necessary for virion assembly (4)(5)(6). Both HDAg-S and HDAg-L possess nuclear localization signals (NLSs) and mainly localize in the nucleus.…”