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標題: | Rta促進Epstein-Barr病毒BRLF1-BZLF1雙基因轉譯的機轉 Promotion of BZLF1 translation by Rta from the BRLF1-BZLF1 bicistronic mRNA of Epstein-Barr virus |
作者: | Sseu-Pei Hwang 黃斯沛 |
指導教授: | 張麗冠(Li-Kwan Chang) |
關鍵字: | EB病毒,Rta,雙基因mRNA,IRES,ITAF, Epstein-Barr virus,Rta,bicistronic mRNA,IRES,ITAF, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | EB病毒 (Epstein-Barr virus, EBV) 為人類皰疹病毒感染全球約90%的人口,在感染人類之後會將病毒基因轉型至宿主細胞內潛伏,使宿主細胞不朽化。雖然感染EB病毒一般並不會造成疾病,但卻可能引發特定癌症發生。潛伏的病毒受到特定外界環境刺激後會使得病毒重新被再活化 (reactivate),由潛伏期 (latency) 轉換至溶裂期 (lytic cycle),製造具有感染性的病毒顆粒。病毒在進行溶裂期複製時首先會表現兩個極早期轉錄因子Rta與Zta,這兩個轉錄因子的能夠進一步開啟病毒其他溶裂期基因表現。表現Rta與Zta的病毒基因BRLF1及BZLF1在病毒基因體上彼此相鄰,能夠由BRLF1基因的啟動子 (Rp) 轉錄出雙基因 (bicistronic) 的BRLF1-BZLF1 mRNA (RZ-mRNA)。過去研究中已經證實Zta蛋白質能夠由RZ-mRNA中被有效的轉譯,而且Rta 蛋白質能夠以cis的方式透過雙基因mRNA中的intercistronic region (ICR) 序列在B淋巴細胞P3HR1細胞株中提升下游BZLF1開放譯讀架的轉譯。本研究發現Rta能夠以trans的方式在293T細胞中活化BZLF1開放譯讀架的轉譯。本研究以建構的雙基因表現質體 (pEGFP-ICR-Luc) 證明Rta為ITAF (IRES trans-acting factor) 能夠反式活化 (trans-activate) 下游基因的表現,且Rta的N端序列為活化時的必要片段。本研究亦對ICR序列進行分段,證明ICR序列中的region I及region II片段對Rta活化雙基因轉譯的重要性。此外本研究也以in vitro RNA-protein pulldown方法證實Rta蛋白質與ICR RNA序列結合。綜合以上結果,本研究證實RZ-mRNA的ICR序列具有IRES活性,而Rta能夠做為ITAF反式活化BRLF1-BZLF1雙基因mRNA中BZLF1開放譯讀區的轉譯,並提升Zta 蛋白質的表現。 Epstein-Barr virus (EBV) is a human herpesvirus, which infects more than 90% of the population. After infection, EBV immortalizes its host cells, establishing latent infection. Although the infection is usually asympotmatic, latent EBV infection is often associated with human cancers. The virus can be reactivated by specific environmental changes and enters the lytic cycle to produce virus particles. At the onset of the lytic cycle, the virus expresses two immediate-early transcription factors, Rta and Zta, which activate EBV’s lytic genes. The genes encoding Rta and Zta, BRLF1 and BZLF1, respectively, are situated adjacent on the viral genome with BRLF1 located upstream. The promoter of BRLF1 (Rp) transcribes a bicistronic BRLF1-BZLF1 mRNA (RZ-mRNA). Earlier study has established that Zta is translated efficiently from the bicistronic mRNA; Rta can elevate the translation of BZLF1 orf located downstream in cis via the intercistronic region (ICR) on the RZ-mRNA in P3HR1 B lymphocytes. This study finds that Rta can trans-activate the BZLF1 translation in 293T cells. By generating a bicistronic reporter plasmid (pEGFP-ICR-Luc), this study verified that Rta acts as an IRES trans-acting factor (ITAF) to activate downstream translation in a trans-acting manner and the N-terminal domain of Rta is essential in the scheme. Deletion analysis of ICR further indicates that the two regions within ICR, region I and region II, are important for the translational activation by Rta. Moreover, RNA-protein pulldown assay confirms that Rta binds to ICR in vitro. Altogether, this study demonstrated that the ICR in RZ-mRNA function as an IRES, while Rta serve as an ITAF that trans-activates the translation of BZLF1 from BRLF1-BZLF1 bicistronic mRNA, thereby promoting the expression of Zta. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71325 |
DOI: | 10.6342/NTU201801775 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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