EB病毒特早期蛋白質Rta活化14-3-3 σ表現及其對 細胞週期之影響

Epstein-Barr virus immediate-early protein Rta activates 14-3-3 σ expression to regulate cell cycle progression

謝旻潔(Min-Jei Hsieh)

指導教授 ： 許翠瑛

國立臺灣大學/醫學院/微生物學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00734

摘要

Epstein-Barr virus (EBV)為人類傳染性單核球增多症及口腔髮狀白斑瘤的病原，亦被發現與許多人類癌症，例如巴氏淋巴瘤(Burkitt's lymphoma)、何杰金氏淋巴瘤(Hodgkin's lymphoma)及鼻咽癌(nasopharyngeal carcinoma, NPC)等有高度相關性。病毒通常會利用本身所產生的蛋白質，影響細胞週期運行所需的相關分子，達到有利於病毒複製的環境。在實驗室先前的研究中，已知EB病毒的特早期蛋白質Rta，具有轉活化p21啟動子之能力，使p21蛋白質表現量上升，p21蛋白質屬於cyclin-dependent kinase inhibitor (CKIs)的一員，Rta藉由轉活化p21啟動子使細胞週期停在G1時期，造成G1-arrest的現象。此外，從Rta cDNA microarray資料顯示，Rta的存在亦活化具有調控細胞週期能力的14-3-3 σ蛋白質。在本研究當中，進一步研究Rta是否會藉由調控14-3-3 σ蛋白質的表現而對細胞週期有所影響。首先，以即時同步偵測定量聚合酶連鎖反應及西方墨點法，確認Rta可在轉錄、轉譯的層面上調控14-3-3 σ蛋白質的表現。接著，欲探討Rta調控14-3-3 σ蛋白質之機制，以螢光酵素-報導基因檢測方法，觀察到Rta具有轉活化14-3-3 σ啟動子的能力，但此轉活化能力並不是透過14-3-3 σ啟動子上的一個putative Rta-responsive element (RRE)。欲觀察Rta調控14-3-3 σ蛋白質是否對細胞週期運行而有所影響，以免疫螢光染色法觀察到Rta可能透過14-3-3 σ蛋白質而影響CDK1、CDK2的入核，使其堆積在細胞質當中，干擾細胞週期的正常運作。此外，Rta亦會造成細胞週期當中G2/M期的遲緩，然而是否是透過14-3-3 σ而造成此現象，還需進一步之探討。在本研究當中，我們發現Rta除了可藉由調控p21之外，亦會藉由影響14-3-3 σ的表現量而共同影響細胞週期的運行。

關鍵字

EB 病毒 ； Rta ； 細胞週期 ； 14-3-3σ

並列摘要

Epstein-Barr virus (EBV) is the etiologic agent responsible for infectious mononucleosis and oral hairy leukoplakia. It is also highly associated with several human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinoma (NPC). Many researches have suggested that regulation of the cell cycle is one strategy frequently used by viruses to create a more favorable environment for viral replication. From the previous studies of our lab, we have demonstrated that EBV Rta had the ability to block cell cycle at the G1 phase through regulating the expression of p21 protein. Furthermore, data from a cDNA microarray indicated that Rta could upregulate 14-3-3 σ in transcriptional level. The 14-3-3 σ protein is reported to interact with many cell cycle-related molecules. In this study, we intend to elucidate whether Rta activates 14-3-3 σ expression to affect cell cycle progression. First, we confirmed that Rta could upregulate 14-3-3 σ mRNA and protein expression by using RT-Q-PCR and western blotting assay. Moreover, we found that Rta could transactivate the 14-3-3 σ promoter in a manner independent of its putative Rta-responsive element. In addition, we demonstrated that Rta affected the subcellular localization of CDK1 and CDK2 in 293-TREx-Rta cells via the upregulation of 14-3-3σ expression. Finally, we discovered that Rta also was able to interfere with the G2-M phase progression of cell cycle. Taken together, our results clearly suggest that EBV protein Rta acts through the transcriptional induction of 14-3-3 σ, in addition to p21, to influence cell cycle progression.

並列關鍵字

Epstein-Barr virus ； Rta ； cell cycle ； 14-3-3σ

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