| Summary: | 博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 95 === Hepatitis delta virus (HDV) is a human pathogen associated with fulminant hepatitis and progressive chronic liver cirrhosis upon superinfection and coinfection with hepatitis B virus (HBV). HDV particles contain a single-stranded circular RNA genome of 1.7 kilobases. The HDV antigenomic RNA contains an open reading frame which encodes two forms of hepatitis delta antigen (HDAg), HDAg-S (24 kDa) and HDAg-L (27 kDa). The two HDAgs are identical in the amino acid sequence, except that the HDAg-L possesses an extra 19-amino-acid extension at the C terminus. Nevertheless, functions of two forms of HDAgs are quite different. The HDAg-S facilitates the replication of HDV RNA, whereas the HDAg-L is required for HDV assembly. Previous studies have also indicated that the HDAg-L contains a nuclear export signal located in its C terminus from amino acid residues 198-210.
To study the molecular mechanisms by which HDV infection causes hepatitis, possible interactions between HDAg-L and host proteins were analyzed. GST pull-down assay was performed with a GST-HDAg-L(198-210) fusion protein and HepG2 cell lysate to identify host proteins that interact with the C terminus of HDAg-L. By performing GST pull-down assay and mass spectrometry analysis, clathrin heavy chain (CHC) that represents the major structural component of the coated pit lattice, was identified to be an HDAg-L(198-210)-interacting protein. In vitro binding assay demonstrated that the N-terminal 107 amino acid residues of the CHC and the putative clathrin box (199-LFPAD-203) in the HDAg-L are critical for the interaction. The specific interaction between HDAg-L and CHC was further demonstrated by confocal microscopy and co-immunoprecipitation. Sucrose gradient centrifugation followed by co-immunoprecipitation analysis indicated that HDAg-L constitutes part of the clathrin complex.
To investigate the biological role of CHC in HDV life cycle, inhibitors of clathrin-mediated transport were used to investigate the association of CHC and HDV assembly. Here, we demonstrated that brefeldin A (BFA) and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly, but had no effect on the assembly of the small surface antigen of HBV. HDAg-L with mutations at the conserved amino acid residues of clathrin box failed to interact with CHC and to assemble into virus-like particles. In addition, small hairpin RNA (shRNA)-mediated CHC down-regulation effectively reduced the assembly of HDV. These data suggest that the formation of HDV VLPs is mainly mediated by the association of HDAg-L with CHC in the trans-Golgi network (TGN), indicating that CHC is a key component to the process of HDV assembly. In addition, possible effects of HDAg-L in regulating the functions of CHC were also examined. In the presence of the surface antigen of HBV, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the ligand-induced degradation of epidermal growth factor receptor in transfected mammalian cells. These data suggest that HDAg-L interferes with the function of clathrin. The cell growth defects observed in HDV-infected hepatocytes may be partially due to a lost of control in clathrin-mediated protein trafficking.
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