蛋白質激酶Cδ在組蛋白去乙醯酶抑制劑誘發EB病毒溶裂期時所扮演之角色

Role of PKCδ in HDACi-Induced Epstein-Barr Virus Reactivation

蔡澤濬(Tze-Jiun Tsai)

指導教授 ： 蔡錦華

國立臺灣大學/醫學院/微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.03224

摘要

EB病毒的生活週期可分為潛伏期及溶裂期兩部份，潛伏期時EB病毒只表現潛伏期基因，且基因體會隨著宿主細胞週期而複製；當EB病毒由潛伏期進入溶裂期時首先會活化BZLF1及BRLF1基因表現，此病毒基因產物可扮演轉活化因子的角色促進其他溶裂期基因表現，使得大量病毒體產生。過去已發現許多刺激會造成EB病毒活化進入溶裂期，包括phorbol ester、抗免疫球蛋白、增加鈣離子通透性、或是組蛋白去乙醯酶抑制劑，關於前三者如何導致EB病毒溶裂期發生已有相當多的研究，但對於組蛋白去乙醯?抑制劑是透過何種機制造成EB病毒溶裂期發生並沒有太多著墨。而在本論文中以西方點墨法透過蛋白質激酶Cδ抑制劑、RNAi、及過度表現無活化型蛋白質激酶Cδ等方式發現蛋白質激酶Cδ在組蛋白去乙醯?抑制劑所誘發之EB病毒溶裂期蛋白表現扮演重要的角色；更進一步的研究顯示組蛋白去乙醯酶抑制劑能夠造成蛋白質激酶Cδ核轉置、酥氨酸505磷酸化增加以及活化片段CF-PKCδ產生的已知之蛋白質激酶Cδ活化標的。而以報導基因檢測發現過度表現蛋白質激酶Cδ會活化EB病毒溶裂期極早期基因BZLF1促進子活性，因此蛋白質激酶Cδ可透過直接或間接的方式調控與BZLF1促進子鍵結之轉錄因子。最後建構不會產生CF-PKCδ以及無法進行核轉置之蛋白質激酶Cδ突變型蛋白表現質體，初步結果顯示完整片段之蛋白質激酶Cδ對於組蛋白去乙醯?抑制劑誘發EB病毒溶裂期訊息傳導較為重要，而無法進行核轉置之蛋白質激酶Cδ則喪失活化EB病毒溶裂期蛋白表現的功能。

關鍵字

蛋白質激酶Cδ ； EB病毒 ； 組蛋白去乙醯酶溶裂期

並列摘要

The life cycle of Epstein-Barr virus (EBV) can be divided into latent and lytic stage. During the latent stage, EBV only express latent genes, and its genome replicates with host normal cell cycle. The switch from latent stage to lytic cycle leads to the expression of two viral genes, BZLF1 and BRLF1. Their protein products have been known to act as transcriptional activators and stimulate other viral lytic genes expression. Activating EBV lytic cycle can be induced by various reagents, including phorbol ester, anti-immunoglobulin, calcium ionophore, and histone deacetylase inhibitor (HDACi). It has been well studied how phorbol ester, anti-immunoglobulin, and calcium ionphore induce BZLF1 gene expression, but the mechanism of HDACi-induced activation of EBV lytic cycle has not been elucidated. In the present study, various tools were utilized to knocken down the ability or the expression level of PKCδ to investigate its role in the HDACi-induced activation of EBV lytic cycle. Upon HDACi treatment, the amount of nuclear PKCδ, the level of threonine 505 phosphorylation, and the production of catalytic fragment PKCδ (CF-PKCδ) were observed to increase. Luciferase reporter assay demonstrated that overexpression PKCδ can stimulate BZLF1 promoter activity. Since rottlerin has little effect on the histone acetylation level on BZLF1 promoter, PKCδ may regulate, directly or indirectly, the activity of transcription factors on BZLF1 promoter. Lastly, we explore the effect of PKCδ mutation on capase-3 cleavage site and nuclear localization signal (NLS). Our preliminary data reveals that the CF-PKCδ production is not required for HDACi-induced activation of EBV lytic cycle, while mutation on PKCδ NLS reduces EBV lytic genes expression.

並列關鍵字

PKCδ ； EBV ； HDAC ； lytic cycle

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