- 學位論文
第一部分: 甘油脂醛-3-磷酸去氫酶與D型肝炎病毒RNA結合在D型肝炎病毒增殖之生物功能研究;第二部分: 細胞因子NESI與大型D型肝炎病毒抗原核輸出訊號之結合及功能分析
Part I: Functional analysis of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binding to HDV RNA in the multiplication of hepatitis delta virus; Part II: Characterization and functional analysis of a cellular factor NESI interacting with the nuclear export signal of large delta antigen
摘要
D型肝炎病毒為一含外套的病毒顆粒，外套蛋白質由B型肝炎病毒之表面抗原所組成。病毒顆粒內部則含有約1.7 kb的單股環狀RNA基因體與其所轉譯的delta抗原。delta抗原可分大型(214個胺基酸；27 kDa)與小型(195個胺基酸；24 kDa)兩者的差別僅在於大型delta抗原的羧端多了19個胺基酸。大小型抗原雖然在胺基酸組成及結構上有相當高的相似性，但兩者在病毒的生活史扮演著不同的角色。大型Delta抗原多出的19個胺基酸對於病毒顆粒的包裹是重要的。其具有促使大型delta抗原由細胞核輸送到細胞質中，與B型肝炎病毒之表面抗原共同組成病毒顆粒之功能。而小型delta抗原為病毒基因體複製所必須，大型delta抗原則抑制之。先前的研究即發現，大型delta抗原C端第198至210胺基酸為一細胞核輸出訊號(nuclear export signal; NES)而被命名為NES(HDAg-L)。本篇論文即針對病毒基因體探討細胞因子GAPDH與HDV基因體結合，對病毒複製的生物功能。另一方面則是針對與delta抗原核輸出訊號結合的細胞因子對病毒生活史的調控。 為了進一步了解GAPDH和HDV RNA間交互作用所扮演的可能功能，以能在哺乳動物細胞中，由SV40 promoter轉錄出HDV雙套體RNA並進行複製的質體pSVD2，轉染Huh7細胞。兩天後以間接免疫螢光染色，以觀察GAPDH在HDV RNA複製情況下，在細胞內分布的情形。在正常狀態下，GAPDH主要位於細胞質中。在轉染細胞中，則可以觀察到部分細胞核中觀測到GAPDH的存在。以間接免疫螢光染色及螢光原位雜交法同時觀察轉染細胞中，HDV RNA所在位置，發現其出現在細胞核質。將包含有GAPDH結合區域的HDV antigenomic RNA片段以活體外轉錄反應方式產生出來；隨著反應時間的增長，在GAPDH存在的狀況下確實使切割產物增加。結果也暗示GAPDH藉由與病毒RNA的結合，而在HDV RNA自我切割之核酸酶(ribozyme)活性扮演重要的角色。 利用酵母菌雙雜交系統我們找到一個會與輸出/包裹功能健全之delta抗原核輸出訊號〈NES(HDAg-L)〉但不與功能缺陷delta抗原核輸出訊號〈NES*(HDAg-L) 〉結合的細胞因子並將之命名為NESI (NES interacting)。NESI主要分佈于人類肝臟組織並包含467個胺基酸，含有一假設的actin結合區及bipartite核位訊息。以共同免疫沉澱及免疫螢光染色法則進一步確認大型delta抗原與NESI的特異性結合。在轉染細胞中過量表現反意(antisense) NESI RNA會抑制由大型delta抗原所媒介之HDV genomic RNA共同包裹於類病毒顆 粒。這些結果指出，藉由與大型delta抗原結合，NESI 發揮其在HDV的包裹扮演著關鍵性的角色。
並列摘要
Heaptitis virus (HDV) is the causative agent of acute and chronic liver diseases including fulminant hepatitis and accelerated liver cirrhosis. HDV particles contain a single-stranded circular RNA genome of 1.7 kilobases. The HDV antigenomic RNA contains an open reading frame which encodes delta antigens( HDAgs). There are two forms of HDAgs, the small HDAg (HDAg-S, 24 kDa) and the large HDAg (HDAg-L, 27 kDa). The two HDAgs are identical in the amino acid sequence, except that the HDAg-L contains an additional 19 amino acids at it C terminus. Nevertheless, functions of two forms of HDAgs are quite different. The HDAg-S is required for the HDV replication, whereas the HDAg-L has negative effects on genome replication is required for HDV assembly. The HDV genome RNA is associated with delta antigen to form ribonucleoprotein. Previous results have shown that a cellular factor, GAPDH interacted with HDV RNA. In addition, nuclear export signal that located in the C-terminus of HDAg-L from amino acid residues 198-210, designated NES(HDAg-L), has been identified. To investigate the biological role of GAPDH in HDV life cycle, cells were transfected with plasmid pSVD2 representing the dimeric HDV cDNA and proceeded to fluorescence in situ hybridization/immunofluorescence staining. In the presence of HDV replication, GAPDH relocalized from the cytoplasm to the nucleus. The biological significance of the interaction between GAPDH and HDV RNA was further elucidate by examing the possible role of GAPDH on the catalysis of HDV ribozyme. This cis-cleavage activity of the HDV anigenomic RNA increase from 34 to 62 % following a 40-min incubation with 50 ng/ l of GAPDH. The enhancing effect of GAPDH on the catalysis of HDV ribozyme was reproducible and was not observed with bovine serum albumin (BSA). These results suggest a possible role of GAPDH involved in HDV multiplication. The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. A previously identified chromosome region maintenance 1 (CRM1)-independent nuclear export signal (NES) at the C-terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) is required for the assembly of hepatitis delta virus (HDV) To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using GAL4 binding domain fused to NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI (NES-interacting protein) that specifically interacted with the wild-type NES(HDAg-L), but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L) in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9 kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of an antisense NESI RNA abolished HDAg-L-mediated assembly of HDV genomic RNA into virus-like particles. These data indicate a critical role of NESI, through the interaction of HDAg-L, involved in the assembly of HDV.
參考文獻
延伸閱讀
- 王永昇（2014）。小型delta抗原與宿主第一型RNA聚合酶共同結合至 D型肝炎病毒基因體特定末端環狀區段之研究〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2014.10663
- Chen, J. Y. (2014). 一、藉由蛋白質體學分析高糖環境下人類肝臟細胞內蛋白質體變化及其參與糖尿病誘導肝臟疾病之相關機制 二、藉由蛋白質體學分析具有pemetrexed抗藥性之人類肺腺癌細胞內蛋白質體變化及參與之相關機制 [master's thesis, National Tsing Hua University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0016-2912201413490912
- 林海寧（2011）。D型肝炎病毒小型delta抗原之中間區段與DNA依賴性RNA聚合酶I之交互作用在病毒複製中扮演的角色〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2011.02637
- An, L. Y. (2017). 探討間葉幹細胞在以N-nitrosodimethylamine誘發成肝炎、肝纖維化及肝癌之大鼠體內的生理、病理角色 [master's thesis, National Taiwan Normal University]. Airiti Library. https://doi.org/10.6345/NTNU202202468
- Hsu, W. Y. (2020). Application of CRISPR/Cas9 genome editing for treatment of HBV infection1. Inactivation of HBV promoters/enhancers by CRISPR/Cas9 genome editing2. Generation of single class I HLA-expressing cell lines for identifying CD8+ T cell epitopes of HBV [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU202003899