探討細胞表面分子CD300a在EB病毒感染中之調控與生物功能

Abstract

EB病毒 (Epstein-Barr virus, EBV) 是具有致癌性的人類皰疹病毒，並且與許多人類惡性腫瘤有高度相關，包含巴氏淋巴瘤 (Burkitt’s lymphoma)、霍杰金氏症 (Hodgkin’s disease)、鼻咽癌 (nasopharyngeal carcinoma, NPC)、移植後淋巴組織增生症 (post-transplant lymphoproliferative disease, PTLD) 等。在體外，EB病毒能夠感染人類初級B細胞 (primary B cells)，並使其轉形成具有持續增生能力之類淋巴母芽細胞株 (Lymphoblastoid cell line, LCL)。 EB病毒為了能長期存活於宿主細胞內，會利用調控宿主之免疫反應，使得EB病毒得以躲避免疫攻擊。本實驗室先前利用cDNA微陣列分析CD19+ 初級B細胞及LCLs，探查兩者細胞內基因表現之變化情形。發現EB病毒感染B細胞後，多種細胞表面標記分子的表現量有受到影響，其中以CD300a的變化量最為明顯。在過去的研究中指出，CD300a為高度保留之基因，並且調控許多免疫細胞，包括抑制NK細胞的胞殺作用、調控漿細胞樣樹突細胞 (pDCs) 內的細胞激素以及抑制肥大細胞的發炎反應等。除此之外，CD300a也與一些疾病以及病毒感染相關，包括在瀰漫型大B細胞淋巴癌 (diffuse large B cell lymphoma, DLBCL) 中維持癌細胞生長以及在登革熱病毒中幫助病毒顆粒進入至宿主細胞內等。另外，CD300a在正常B細胞中表現量是極少的，但在EB病毒感染之B細胞中表現量卻有明顯上升的現象，而實際上EB病毒與CD300a之間的關係目前並不清楚。因此本研究目的是為了瞭解CD300a於EB病毒感染B細胞中之調控機制及細胞功能。實驗結果發現，隨著EB病毒感染B細胞的天數增加，CD300a表現量也有隨之上升的現象。另外在七位健康捐血者的B細胞以EB病毒感染後不朽化成的LCLs中也能看到CD300a皆有大量表現，顯示EB病毒具有提升CD300a表現的能力，且此一現象是存在於所有EB病毒感染後的LCLs中。進一步，發現EB病毒的潛伏期核蛋白1 (Epstein-Barr Virus nuclear antigen 1, EBNA1) 及潛伏期核蛋白2 (Epstein-Barr Virus nuclear antigen 2, EBNA2) 對於誘發CD300a的mRNA及蛋白質是重要的。為了瞭解EBNA1及EBNA2是如何調控CD300的表現，進一步利用軟體分析CD300a啟動子上可能含有之相關轉錄因子結合位，發現有與EBNA1相關之AP-1結合位以及與EBNA2相關之RBP-Jκ結合位，並以螢光酵素報導基因分析顯示，CD300a啟動子上 -1498至-208片段上的AP-1結合位對於EBNA1活化CD300a表現是重要的，而 -208至-145片段上的RBP-Jκ結合位則是對於EBNA2活化CD300a表現是重要的。除此之外，在LCLs中以shRNA抑制RBP-Jκ表現後，發現CD300a表現量有減少的現象，暗示EBNA2可能透過與RBP-Jκ之結合來調控CD300a表現。 進一步，欲瞭解CD300a在EB病毒感染宿主細胞中所扮演之生物角色及生理功能。發現在LCLs中以shRNA抑制CD300a表現後，觀察到EB病毒溶裂期基因的表現量皆有上升的現象，顯示LCLs內EB病毒進入溶裂期。由於EB病毒進入溶裂期後會先表現溶裂期極早期基因Rta及Zta，為了進一步探討CD300a是如何抑制EB病毒進入溶裂期，因此利用Rta啟動子 (Rp)及Zta啟動子 (Zp) 以螢光酵素報導基因分析，結果顯示CD300a可能藉由抑制Rp的方式進而抑制EB病毒進入溶裂期。除此之外，在過去的報導中指出CD300a具有調控細胞激素的能力，因此進一步探查是否在EBV感染之細胞中也有相同的功能。發現在LCLs中以shRNA抑制CD300a表現後觀察到IL-6有活化的現象，但TNF-α則沒有太大的影響，顯示CD300a在EBV感染之細胞中同樣具有影響細胞激素表現的能力。進一步，欲探查抑制CD300a之LCLs命運為何，同樣在LCLs中以shRNA抑制CD300a表現後，則發現會減少細胞增生的速率，但不影響細胞週期，暗示CD300a能幫忙維持EB病毒感染之細胞存活。 總結以上，EB病毒透過EBNA1及EBNA2調控CD300a表現，維持宿主細胞之存活，並且幫助其長期潛伏於宿主細胞中。

Epstein-Barr virus (EBV) is a human oncogenic herpesvirus and its infection is associated with many human malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, Post-transplant lymphoproliferative disorder etc. In vitro, EBV can immortalize primary B cells into unlimitedly proliferating lymphoblastoid cell lines (LCLs). In order to persist in host cells, EBV manipulates the host immune response so that it can escape the immune surveillance. Previously, we utilized cDNA microarray to compare the whole panel of cellular gene expression between CD19+ primary B cells and LCLs immortalized with B95.8 strain EBV. Preliminarily, we found that EBV influences the gene expression of several cluster of differentiation markers (CD markers), especially CD300a. Previous study showed that CD300a is a highly conserved gene and can regulate different immune cells, such as inhibiting the killing activity of NK cells, regulating cytokines expression of pDCs, inhibiting the inflammatory response of mast cells etc. In addition, CD300a is also associated with different diseases and viral infection, such as maintenance of tumor cells survival in DLBCLs, assisting the infection of dengue virus etc. The expression level of CD300a is very low in normal B cell but is significantly upregulated in EBV infected B cells. So far, the correlation between EBV and CD300a is still unknown, so we want to find out the role of CD300a in EBV infected cells. The expression of CD300a is upregulated during EBV infection. B cells purified from seven different donors are infected by EBV for 28 days (defined as LCLs) and CD300a is upregulated in these LCLs. Next, we would like to know which viral gene is involved in these upregulation effects. We found that the Epstein-Barr Virus nuclear antigen 1 (EBNA1) and Epstein-Barr Virus nuclear antigen 2 (EBNA2) are responsible for inducing CD300a expression in both mRNA and protein levels. To understand how EBNA1 and EBNA2 regulate CD300a expression, we predict the promoter region of CD300a. We found a AP-1 binding site, which is the binding site of EBNA1 and a RBP-Jκ binding site, which is the binding site of EBNA2. Based on the result of luciferase reporter assay, we demonstrated that the AP-1 binding site located at the -1498 to -208 locus of CD300a promoter is crucial for EBNA1 upregulating CD300a. However, the RBP-Jκ binding stie located at the -208 to -145 locus of CD300a promoter is crucial for EBNA2 upregulating CD300a. In addition, knockdown of endogenous RBP-Jκ in LCLs with shRNA suppresses the CD300a expression. These data implied that EBNA2 upregulates CD300a through RBP-Jκ. Next, we desired to address the influence of this upregulation in EBV infected cells. Knockdown of CD300a in LCLs induces EBV lytic protein expression. Since Rta and Zta are the immediate early genes expressed in lytic cycle. We use Rta promoter (Rp) and Zta promoter (Zp) in luciferase reporter assay. This result shows that CD300a suppresses EBV lytic cycle by suppressing Rp. In addition, previous study showed that CD300a can regulate cytokines expression. Knockdown of CD300a in LCLs also induces the expression of IL-6 instead of TNF-α. These data indicate that CD300a can affect particular cytokine production. To elucidate the fate of CD300a depleted LCLs, we use shRNA to deplete CD300a. We found that knockdown of CD300a reduces cell proliferation but does not impact cell cycle. These result imply that CD300a can help cell survival in EBV infected cells. To sum up, both EBNA1 and EBNA2 can regulate CD300a so that EBV can prevent host cells from program cell death and persist in host cells.