探討EB病毒溶裂期轉活化子Zta誘發細胞內Egr-1之基因表現

Abstract

EB病毒的生活史中存在兩種狀態：潛伏期和溶裂期。根據過去的研究，當EB病毒進入溶裂期時可能造成細胞的改變。為了解溶裂期進行時對於細胞環境的影響，我們實驗室使用微矩陣分析法偵測先前建立的293A潛伏期與溶裂期細胞株中，並比較細胞基因表現之差異。結果發現，相較於潛伏期細胞，溶裂期細胞中可以偵測到較高量的Egr-1基因產物。利用西方墨點分析實驗，證實溶裂期細胞中Egr-1蛋白質質表現確實高於潛伏期細胞中。另一方面，在使用針對Zta專一性之siRNA表現質體(siZ1)抑制溶裂期發生時，即無法偵測到Egr-1蛋白質質量增高的現象。據此，推測當細胞中的EB病毒進入溶裂期時可引發Egr-1蛋白質質表現量上升。為進一步探討Egr-1蛋白質質量之增加是否受到病毒溶裂期基因產物的影響，利用單一基因轉染的方式將溶裂期病毒蛋白質質表現於不含EB病毒的細胞株中，並偵測Egr-1蛋白質質之表現。由實驗結果得知，在NPC-TW01和NPC-TW04細胞中，Zta蛋白質質可明顯促進細胞中Egr-1蛋白質量增加。同樣地，在先前實驗室所建立之Zta誘發性細胞株(HONE-1-52)也觀察到相同結果。進一步，利用報導質體實驗分析，也能證實Zta可以轉活化Egr-1啟動子之活性。根據以上結果得知，Zta蛋白質可以利用轉錄層級的機制調控Egr-1基因，並促進其蛋白質之表現。為了解Zta啟動Egr-1基因的詳細機制，利用一系列Egr-1啟動子的刪除突變報導質體進行分析，發現啟動子上–503bp~–438bp和–438bp~–395bp的區域對於Zta活化Egr-1啟動子而言是重要的。根據過去文獻記載以及利用軟體分析後知道-452bp~-438bp區域中存在兩個可能的ZREs (Zta response elements)，而-431bp~-407bp間存在一Elk-1/SRF結合位置。另一方面，由過去研究Egr-1基因的調控，已知細胞受到血清或者生長因子的刺激下，可以藉由活化的Ras/Raf/Mek/Erk/Elk訊息路徑啟動Egr-1基因表現。有趣的是，在HONE-1-52細胞中，Zta確實可使Erk蛋白質的磷酸化的程度增加。而加入Erk抑制劑時，即使Zta蛋白質表現也無法有效活化Egr-1基因，代表Erk的磷酸化對於Zta啟動Egr-1基因而言相當重要。綜合以上結果，EB病毒溶裂期轉活化子Zta可能藉由直接與啟動子之結合以及間接活化Erk訊息誘發細胞中Egr-1基因之表現。然而，就目前為止，Zta所引發的Egr-1蛋白質表現其生物功能仍然未知，需要日後投入更多的研究工作以探討之。

Epstein-Barr virus (EBV) contains two phases in its life cycle: latent and lytic stages. According to previous studies, the progression of lytic cycle may affect many cellular events. To address this issue, we performed a microarray analysis on two groups of EBV-infected 293 cells that were distinguished into latent and lytic clones. We found that the expression of early growth response-1 (Egr-1), a zinc finger transcription factor, was increased in lytic clones, but not in latent clones. Immunoblot analysis confirmed that Egr-1 protein expression was associated with EBV lytic cycle. Zta is an immediate-early protein encoded by BZLF1 gene and plays a significant role to initiate viral lytic cascades. By using BZLF1-targetd siRNA to block EBV lytic progression, the expression of Egr-1 protein was reduced. Therefore, we suggested that the expression of Egr-1 protein was the downstream event of EBV lytic cycle. Furthermore, by using single gene transfection assay, we identified that Zta could induce Egr-1 protein expression in two EBV-negative cell lines, NPC-TW01 and NPC-TW04. In addition, in a Zta-inducible cell line (HONE-1-52), Egr-1 protein was also induced during Zta expression. As the regulator of EBV lytic cycle, Zta also affects the cellular gene expression at transcriptional level either by directly binding to a consensus DNA sequence or indirectly activating cellular signaling pathways. According to the reporter assays, Zta could activate Egr-1 promoter. By analysis of the deletion mutants of Egr-1 promoter, two regions relative to the transcriptional start site, -503bp~-438bp and –438bp~-395bp, were proven to critical for Zta-mediated activation. These two promoter regions contained putative ZREs (Zta response elements) and Elk-1/SRF binding site, respectively. The Elk-1/SRF binding site was crucial for Egr-1 gene expression upon Ras/Raf/Mek/Erk/Elk signaling pathway triggered by serum and many growth factors. Interestingly, Zta could dramatically increase the phosphorylattion status of Erk-1 protein in our study. In the presence of Erk phosphorylation inhibitos, PD98059 and U0126, Zta-induced Egr-1 protein expression was decreased significantly. Taken together, we demonstrate that the EBV lytic transactivator Zta can induce the expression of cellular Egr-1 gene, and the mechanism is dependent on direct DNA binding and indirect Erk signal activation. The biological meanings of Zta-induced Egr-1 protein remain be investigated in future study.