探討與D型肝炎抗原造成細胞及斑馬魚生長遲緩之宿主基因

Abstract

D型肝炎病毒(hepatitis delta virus, HDV)為一具有外套之球狀病毒顆粒，外套蛋白質由B型肝炎病毒表面抗原所組成，其內部組成為一單股環狀長約1.7 kb之RNA基因體及其所轉譯之delta抗原。Delta抗原有兩種：大型delta抗原(HDAg-L; 214個胺基酸，約為27 kDa)及小型delta抗原(HDAg-S; 195個胺基酸，約為24 kDa)。以功能上而言：大型delta抗原具有與B型肝炎表面抗原組成類D型肝炎病毒顆粒之能力，而小型delta抗原則為病毒RNA基因體複製所必需。比較胺基酸序列發現：大型delta抗原在C端比小型delta抗原多出19個胺基酸（第196到214胺基酸），並且發現此段含有細胞核輸出訊號（nuclear export signal；NES）之功能。 根據以前的研究報告指出，大量表現delta抗原發現有細胞毒殺現象，並且利用baculovirus感染的昆蟲細胞系統，證明delta抗原表現會造成cell cycle arrest。本論文目的為釐清delta抗原造成的細胞毒殺及細胞停止生長的現象，利用流式細胞儀分析轉染短暫及持續表現大型和小型delta抗原的細胞，發現在有大型delta抗原表現的細胞其細胞週期在sub-G0增加許多，但小型delta抗原表現的細胞則與對照組沒有明顯差異。同時也發現大型delta抗原表現的細胞其生長速率會降低。因此，推測大型delta抗原C端的19個胺基酸可能是影響生長遲緩及細胞週期變異的主要因素。此外，以微陣列分析（Microarray），找出與細胞生長相關之基因，並發現這些基因與細胞內p53蛋白質相關。進一步研究得知delta抗原可以抑制p53的轉錄，以減少p53蛋白質的產生，並減少p53在第15號位置之磷酸化。進一步研究證明delta抗原對p53蛋白質所調控之下游基因GADD45A之mRNA含量亦有抑制的情形。 近年來斑馬魚（Zebrafish, Danio rerio）發展為一研究脊椎動物胚胎發育的重要模式動物，其胚胎發育上的機制與哺乳動物是非常相似。因此，以微注射（microinjection）的技術將帶有大型及小型delta抗原之表現質體分別打入單細胞時期之受精卵，以建立基因轉殖魚之動物模式。根據觀察結果發現正常卵發育至可游動之魚體需2~3天，而帶有大型或小型delta抗原之卵其發育需延遲至4~5天才可完全發育成可游動之魚體（swimming larva），並約至第9天開始死亡，推測delta抗原影響斑馬魚之發育及成長過程。本論文提供哺乳細胞及動物模式系統以探討delta抗原如何影響參與D型肝炎致病機轉之研究平台。

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus that provides surface antigens (HBsAg) for assembly and production of infectious virus particles. Inside HDV particles, HDV genomic RNA comprises a negative, single-strand circular RNA molecule of 1.7kb that is associated with delta antigens (HDAgs). There are two forms of delta antigen, the small HDAg (HDAg-S, 24 kDa) and the large HDAg (HDAg-L, 27 kDa). The HDAg-S is essential for the replication of HDV RNA whereas HDAg-L is required for viral assembly. The two HDAgs are identical in the N-terminal 195 amino acid residues, but there is an extra 19-amino-acid extension at the C terminus of the HDAg-L that include a nuclear export singal and an isoprenylation motif.. Overexpression of HDAgs was previously demonstrated to be cytotoxic to host cells In addition, cell cycle arrest was observed in insect cells infected with HDAg recombinant baculovirus. In this study, the effects of HDAgs on cell growth and cytotoxity were examined. Results from flow cytometry analysis revealed an increase of the population of subG0 phase in HDAg-L-expressing cells, but there was little effect in HDAg-S-expressing cells when compared with that of parental cells. In addition, stable clones that express HDAg-L grew at a lower rate. Thus, the unique C-terminus of HDAg-L could be the effecter of cell growth retardation and cell cycle arrest. Host genes associated with cell growth, differentiation, and apoptosis in HDAg-expressing cells were examined by genomic analysis. Several genes associated with p53-related pathways were identified to be differentially expressed in HDAg-expressing cells. Further studies demonstrated that both HDAg-L and HDAg-S inhibited the expression of p53 at transcription and translation level. In addition, the phosphorylation of p53 at Ser15 was decreased in the HDAg-L-expressing cells. Furthermore, the mRNA level of the downstream gene GADD45A was inhibited by delta antigens. Due to the similarity of the mechanisms of embryonic development in zebrafish and mammals, zebrafish currently used as a model system for the studies of developmental biology in vertebrate, To learn the pathogenesis of HDAgs, a zebrafish model was established in this study by microinjection independently into embryos at one-cell stage with plasmids encoding HDAg-L and HDAg-S. Following microinjection, control embryos grew up and became swimming larvae in 2~3 days. Nevertheless, embryos microinjected with HDAg-L and HDAg-S could not develop into swimming larvae until 4~5 days, and the larvae died after the 9th day. It is presumed that delta antigens play important roles in embryonic development and growing up. These studies provided an animal model system to examine the molecular mechanisms of delta antigens involved in the pathogenesis of hepatitis delta virus.