EB病毒蛋白質激酶BGLF4 轉譯調控機制之探討

Translational Regulation of EBV BGLF4 Protein Kinase

陳坤良(kun-Liang Chen)

指導教授 ： 陳美如

國立臺灣大學/醫學院/微生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10382

摘要

BGLF4是EB病毒在進入溶裂期時表現的serine/threonine蛋白激酶，目前已知BGLF4的受質包含有：BMRF1、Zta、EBNA-LP、EF-1δ等。已知BGLF4存在於病毒DNA複製區以及成熟病毒顆粒中，推測BGLF4在病毒的複製過程以及殼體化（encapsidation）中扮演重要的角色。我們實驗室先前利用5’RACE在BGLF4開放譯讀架上游201 bp和255 bp的位置找到其轉錄起始位點。而在本篇論文中除了先前已知的轉錄起始點外，我們在BGLF4開放譯讀架上游472 bp位置上發現另一轉錄起始點。而對於BGLF4長片段的5’端未轉譯區，我們進一步的探討是否具有內部核糖體進入位點（Internal Ribosome Entry Site , IRES）的調控機制。藉由雙譯讀架（bicistronic reporter）DNA以及RNA短暫轉染至293T細胞中證明BGLF4的5’端未轉譯區含有IRES的活性。另外本篇論文中發現BGLF4基因的IRES活性會受到內質網壓力(ER Stress)反應刺激增加，同時我們在EBV positive Akata細胞中觀察到EB病毒的再活化（reactivation）會使細胞產生ER Stress。而在NA細胞中，當利用Rta刺激EB病毒進入溶裂期後BGLF4基因IRES活性會因為EB病毒進入溶裂期而增加。表示BGLF4 5’端未轉譯區上的IRES對於EB病毒在進入溶裂期時能夠幫助BGLF4蛋白激酶的表現。

關鍵字

EB病毒

並列摘要

Epstein-Barr virus (EBV) BGLF4 encodes the serine/threonine protein kinase which can phosphorylate several viral substrates including BMRF1, Zta, EBNA-LP and cellular translation factor EF-1d. BGLF4 was detected in viral DNA replication compartment and mature virion, suggesting it may play roles in regulating virus DNA replication and encapsidation or egress process. In our previous study, two transcription start sites at -255 and -201 upstream of translation initiation site were identified in 5’-RACE analysis. In this study, we used another primer sets to further identify another transcription start sites of BGLF4 at -472 nt, which is 25 nt downstream of a putative TATA box. To examine whether the long 5’ untranslated region (UTR) of BGLF4 might function as the internal ribosome entry site (IRES), dual luciferase reporter assay was performed with DNA or RNA transfection to demonstrate the translation enhancement effect of BGLF4 5’-UTR. We further demonstrated the BGLF4 IRES activity was enhanced under tunicamycin treatment which would induce the ER stress. Additionally, induction of ER stress was observed upon EBV reactivation in anti-IgG induced EBV positive Akata cells as revealed by the detection of the spliced form of XBP-1. Simultaneously, BGLF4 IRES activity was enhanced upon EBV reaction induced by Rta in EBV-positive NA cells. Data here thus suggest the 5’-UTR of BGLF4 may ensure BGLF4 expression while the cap-dependent translation machinery is blocked in lytic virus replicating cells.

並列關鍵字

BGLF4 ； IRES

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