Clathrin依賴性之蛋白質運輸路徑參與D型肝炎病毒之組合及致病機轉

Abstract

D型肝炎病毒(HDV)為一具有外套之球狀RNA病毒，其外套蛋白質由B型肝炎病毒之表面抗原所組成，並藉此感染肝細胞。病毒顆粒內部則含有約1.7 kb的單股環狀RNA基因體，其所轉譯產生之delta抗原有兩種：大型delta抗原（214個胺基酸，約為27 kDa）和小型delta抗原（195個胺基酸，約為24 kDa）。比較大小型delta抗原的胺基酸序列可以發現，兩者的差別僅在於大型delta抗原的羧端多了19個胺基酸；而在功能上的比較則可以發現，小型delta抗原為病毒RNA基因體複製所必需，而大型delta抗原具有與小型B型肝炎表面抗原組成類D型肝炎病毒顆粒之能力，且C端所多出的19個胺基酸對於病毒顆粒之包裹是重要的，具有促使大型delta抗原由細胞核輸送到細胞質中，與B型肝炎表面抗原共同組成病毒顆粒之功能。並且之前的研究也證明，大型delta抗原C端之第198至第210胺基酸具有細胞核輸出訊號（nuclear export signal；NES）之功能。 為了進一步了解HDV感染所造成的致病原因，首先，分析大型delta抗原C端與宿主細胞因子之間的交互作用，利用GST pull-down assay研究發現，一細胞因子clathrin heavy chain蛋白質(CHC)可能直接或間接對於大型delta抗原的細胞核輸出訊號有專一性的辨認。仔細比較大型delta抗原C端的胺基酸序列，我們發現大型delta抗原第199至203的胺基酸序列（199-LFPAD-203），與clathrin box的序列具有高度相似性。並且進一步利用點突變（L199A及D203A）的實驗，證明大型delta抗原的確利用其C端clathrin box的序列，與CHC結合。以in vitro蛋白質親和力之結合試驗及in vivo共同免疫沉澱的方式，更證實CHC與大型delta抗原C端具有專一性結合的現象。以共軛焦顯微鏡觀察，在小型B型肝炎病毒表面抗原存在下，CHC與大型delta抗原在細胞中的共同座落的位置為trans-Golgi network (TGN)中。在蔗糖梯度離心實驗中，也證實大型delta抗原與CHC存在於同一個複合體中。 Clathrin是細胞中主要進行vesicles形成和運送之重要蛋白質，利用clathirn所調控之胞外分泌(exocytosis)及內涵體運輸(endosomal trafficking)的抑制劑，brefeldin A及wortmannin，可以有效抑制HDV類病毒顆粒之組裝及釋出，但對小型B型肝炎病毒表面抗原之釋出並無明顯影響。將大型delta抗原之clathrin box進行點突變，也發現HDV類病毒顆粒之組裝及釋出有明顯降低的結果；另外，利用shRNA降低細胞內生性CHC之表現，對於HDV類病毒顆粒包裹也有抑制的結果，因此可以推論CHC對HDV病毒包裹的重要性。在哺乳類細胞的系統中，已知運鐵蛋白(transferrin)的內吞作用(endocytosis)及上皮生長因子(EGF) 之降解作用(degradation)也是依靠clathrin所調控的，因此，我們也證明大型delta抗原C端與CHC的結合，會影響運鐵蛋白的內吞或運輸作用；另外，也證明了細胞內上皮生長因子之降解作用，可能因細胞質內大型delta抗原的存在而有減緩的現象，推測CHC與大型delta抗原C端的專一性結合，會影響到細胞內正常依賴clathrin進行的蛋白質之運輸作用。 本論文顯示大型delta抗原為一clathrin adaptor-like的蛋白質，並進一步瞭解大型delta抗原與clathrin的交互作用可能參與在D型肝炎病毒之病毒分泌及其致病機制。

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.