探討雙重特異性去磷酸酶在EB病毒感染細胞中之角色

Abstract

EB病毒(Epstein-Barr virus，EBV)為人類第四型皰疹病毒，是一種帶有外套膜的雙股DNA病毒，主要感染人類B淋巴球細胞，此外也會感染上皮細胞和T淋巴球細胞。EB病毒具有將B淋巴球細胞轉形成為不朽的淋巴母細胞株之能力，在EB病毒感染初級B淋巴球細胞並將之轉形的過程中，會誘發許多訊息傳遞路徑的活化，像是NF-κB，PI3K及MAPK訊息傳遞路徑等等。 先前實驗室經由cDNA微陣列分析EB病毒感染CD19+初級B淋巴球細胞後細胞中基因的表現變化，發現一群隸屬於DUSPs(dual-specificity phosphatases)家族的去磷酸酶的表現量會有所差異。DUSPs隸屬於酪胺酸去磷酸酶(PTP)家族的一員，可以同時針對同一個受質上的磷酸化色胺酸或磷酸化酥胺酸(phospho-Ser/ phospho-Thr)和磷酸化酪胺酸(phosphor-Tyr)上進行去磷酸化作用。DUSP家族中有許多成員被報導對於MAPK訊息傳遞路徑具有特異性負向調控的功能。 在此研究中，我們進一步利用即時定量聚合酶連鎖反應(real-time PCR)與反轉錄聚合酶連鎖反應(RT-PCR)檢驗，證實利用B95.8 EB病毒株感染自白血球濃厚液中純化出來的CD19+ B細胞後，被EB病毒感染之細胞內的某些DUSPs表現量的確有顯著變的趨勢。本研究發現許多DUSPs像是DUSP1、DUSP5、DUSP6、DUSP7、DUSP8、DUSP16、DUSP19和DUSP22，它們在細胞中的表現量會隨著病毒感染和轉形的過程而有降低的趨勢，這暗示著EB病毒除了透過自身的病毒基因如LMP1和LMP2A正向調控MAPK訊息傳遞路徑之外，或許也透過了另一條負向調控的路徑來協助MAPK訊號得以在轉形的過程中持續活化。 此外，我們也發現在淋巴母細胞株中轉染DUSP1或DUSP6基因會影響淋巴母細胞株中的生長與凋亡，這或許反映了為何在轉形過程中這些DUSPs基因的表現需要受到調控。然而，關於EB病毒究竟如何調控細胞中DUSPs表現的機制則尚待更進一步地探討。

Epstein-Barr virus (EBV) is a oncogenic gamma-herpesvirus that persistently infects over 90% of the human population. EBV-encoded genes have been shown to be involved in immune evasion and in the regulation of various cellular signaling cascades, including MAPK, PI3K/AKT and NF-κB pathway. Dual-specificity phosphatases (DUSPs) are a subset of protein tyrosine phosphatases, that can dephosphorylate both phosphotyrosine and phosphoserine/ phosphothreonine residues within the one substrate. DUSP family is proposed as a set of molecular control devices specifying and modulating MAPK signaling, the regulated expression and activity of DUSP family members in different cells and tissues controls MAPK intensity and duration to determine the type of physiological response. Previous research indicates that many cellular gene expressions are altered after EBV infection. By cDNA microarray analysis, we found that several DUSPs expression would be changed during EBV infection in human CD19+ B cells, such as DUSP1、DUSP5、DUSP6、DUSP7、DUSP8、DUSP16、DUSP19 and DUSP22. Further study by Q-PCR and RT-PCR shows that DUDP1 and DUSP6 would significantly down-regulated in EBV-transformed lymphoblastoid cell lines (LCLs). Further study shows that, ectopic expression of DUDP1 and DUSP6 would result in down-regulation of cell proliferation, but not alteration of cell cycle distribution. However, the detail correlation between EBV and DUSPs need further research to investigate.