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標題: | 小型delta抗原抑制核醣體RNA之新合成 Small delta antigen inhibits de novo synthesis of ribosomal RNA |
作者: | Mei-Chen Huang 黃玫甄 |
指導教授: | 張明富(Ming-Fu Chang) |
關鍵字: | 小型delta抗原,核醣體RNA, small delta antigen,ribosomal RNA, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | D型肝炎病毒 (hepatitis delta virus,HDV) 為一具有外套之球狀病毒顆粒,為B型肝炎病毒之衛星病毒,需藉由B型肝炎病毒表面抗原組成其外套才具有感染宿主之能力。其內部由一單股、負向、環狀封閉,長約1.7 kb之RNA基因體及所轉譯之蛋白質delta抗原所組成。D型肝炎病毒的delta抗原有兩種:小型delta抗原 (HDAg-S;195個胺基酸,約24 kDa) 及大型delta抗原 (HDAg-L;214個胺基酸,約27 kDa)。小型delta抗原為病毒RNA基因體複製所必需,而大型delta抗原主要是負責參與病毒顆粒之包裹。
目前對於D型肝炎病毒複製之詳細機轉尚未完全瞭解,但有愈來愈多證據暗示D型肝炎病毒的genomic RNA與antigenomic RNA可能是透過宿主細胞中不同的RNA聚合酶來進行複製。有研究發現HDV antigenomic RNA之合成不受α-amanitin所抑制,而本實驗室發現delta抗原與核仁素 (nucleolin) 結合,分佈於核仁之現象對於D型肝炎病毒的複製是重要的,因此我們推測antigenomic RNA之合成可能是透過RNA polymerase I來進行,同時delta抗原在其中扮演一重要角色。先前本實驗室在in vivo及in vitro實驗中皆證實了小型delta抗原會與RNA polymerase I最大次單元RPA194有直接的交互作用。另一方面,利用reporter assay亦發現在大量表現小型delta抗原的細胞中,小型delta抗原會抑制rRNA基因promoter之活性。 在本論文中利用共同免疫沈澱法再次證明小型delta抗原會與RPA194結合,而且這樣的交互作用可以直接在細胞核萃取物中偵測得到,顯示小型delta抗原與RNA polymerase I之結合在細胞核中是存在的。另外,利用反轉錄-即時定量聚合酶連鎖反應 (RT real-time PCR) 分析發現,小型delta抗原會降低宿主細胞中 ribosomal RNA之新合成 (de novo synthesis),尤其在持續表現小型delta抗原的穩定細胞株中尤為明顯。但若是表現無法分佈至核仁之突變型小型delta抗原,則不具有抑制rRNA基因promoter活性之能力,暗示小型delta抗原對rRNA基因轉錄之抑制是直接作用於核仁中的。此外,為瞭解RNA polymerase I是否真的參與HDV RNA之複製,我們利用核糖核蛋白質複合體免疫沈澱法 (ribonucleoprotein immunoprecipitation assay,RIP assay) 進行實驗,初步發現RNA polymerase I能與HDV genomic RNA共同沈澱下來。以上結果支持RNA polymerase I可能參與D型肝炎病毒antigenomic RNA之複製。 Hepatitis delta virus (HDV) is a spherical enveloped virus. It is a satellite virus that requires hepatitis B virus as its helper virus to provide the envelope proteins important for natural transmission. The nucleocapsid of HDV consists of a single-stranded, negative sense, circular RNA genome of 1.7 kb and the only known encoded protein, delta antigen. There are two forms of delta antigen, the small delta antigen (HDAg-S, 195 amino acids, 24 kDa) and the large delta antigen (HDAg-L, 214 amino acids, 27 kDa). The HDAg-S is required for the replication of HDV RNA, whereas the HDAg-L is required for the viral assembly. The detailed mechanisms of HDV replication are still unclear. However, accumulated evidences suggest that the replication of the genomic and antigenomic RNAs involve two independent transcriptional machineries. The synthesis of antigenomic RNA was demonstrated to be resistant to α-amanitin. In addition, our laboratory has previously demonstrated that HDAg-S bound to nucleolin and localized to the nucleolus at the early stage of viral replication. Therefore, nucleolus- localized RNA polymerase I is a potential candidate for the replication of HDV antigenomic RNA. Further studies in our laboratory demonstrated that HDAg-S interacted with the largest subunit of RNA polymerase I, RPA194, both in vivo and in vitro. In addition, overexpression of HDAg-S inhibited the promoter activity of rRNA genes. In this study, the association between HDAg-S and RPA194 was further confirmed in the nuclear extract, indicating that HDAg-S interacts with RPA194 in the nucleus. In addition, the synthesis of rRNA was markedly decreased in both cells transiently and stably expressing HDAg-S. On the other hand, a nucleus-localized mutant form of HDAg-S that was excluded from the nucleolus fails to inhibit the promoter activity of rRNA genes. These data suggested that the HDAg-S directly inhibits the de novo synthesis of rRNA in the nucleolus. Ribonucleoprotein immunoprecipitation assay was further demonstrated an association of RNA polymerase I with HDV genomic RNA. Taken together, these data indicate that RNA polymerase I may involve in the synthesis of HDV antigenomic RNA. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32746 |
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顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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