EB病毒裂解性轉錄活化子Rta之結構解析

Abstract

EB病毒也被稱為人類泡疹病毒第四型，為雙鏈DNA病毒，是最常引起人類疾病的病毒之一，估計全世界超過95%的人口皆曾受其感染。原發性的EB病毒感染通常從口腔開始，透過唾液傳播來感染CD21+ B淋巴細胞及上皮細胞。EB病毒感染會造成傳染性單核白血球增多症，並可能導致多種惡性腫瘤的發生，目前已知伯基特淋巴瘤、霍奇金淋巴瘤、胃癌及鼻咽癌等皆與此病毒相關。EB病毒的複製週期可分裂解性複製型或潛伏型兩種型式，一旦病毒入侵宿主細胞後就會採取其中一種型式進行後續的感染。雖然這些惡性腫瘤的成因大多源自於EB病毒潛伏感染所引發的NF-κB路徑活化，不過在惡性腫瘤中也有少數病毒會經由再活化的過程轉而以裂解性方式進行複製，此時細胞會分泌生長因子和致癌細胞因子，促使腫瘤惡化及轉移。因此為了更清楚知道EB病毒感染的風險，有必要更深入的了解病毒如何調控潛伏型和裂解性複製之間的轉換。 EB病毒的裂解性複製包含三個時期，為立即早期 (IE)、早期 (E)及晚期 (L)，其中立即早期會藉由表現BZLF1和BRLF1基因來合成轉錄活化子Zta和Rta，二者共同調節下游之病毒與宿主基因的表達，以利病毒基因的複製。而當感染EB病毒的細胞接受到細胞因子的刺激時，也可能引發BZLF1或BRLF1基因的活化，促進EB病毒由潛伏轉換成裂解性複製，因此Zta和Rta實為啟動EB病毒再活化的關鍵。Rta可以直接與Rta響應元件 (RRE)結合，或透過和Zta的協同作用調控基因表達，亦可以藉由間接的方式，和作為媒介的蛋白MCAF1作用後，與其他轉錄因子的促進子結合， Rta活化本身及Zta即是以這種方式達成。 雖然Zta及Rta都是轉錄活化子，但它們的功能及DNA結合特性皆有明顯差異。目前對於Rta結構與功能的了解仍然十分有限。由於在腫瘤細胞中執行EB病毒裂解誘導被視為一種具有潛力的治療策略，鑒於Rta導致的EB病毒再活化是病毒傳播的主要機制，所以有必要更詳細地了解Rta。因此本研究的主要目標就是要解析Rta或是Rta-DNA複合體的結構，以此來闡述Rta功能。 目前已經成功在BL21-star菌株中表現6x His-tag的Rta，藉由鎳離子親和性、肝素瓊脂及分子篩管柱層析法進行純化蛋白，可以得到均質狀且構型正確的Rta同源二聚體，並透過凝膠遷移實驗確認其仍然具有與特定DNA序列結合的能力。我亦嘗試使用多種養晶試劑以培養Rta同源二聚體的蛋白晶體，同時也使用BMLF1-RRE來形成Rta-DNA複合體，並進行蛋白晶體培養。目前已發現複合體可能的結晶條件，後續實驗將針對此條件進行優化，以取得高品質的Rta-DNA晶體進行後續的結構解析。

Epstein–Barr virus (EBV), also known as human herpesvirus 4 (HHV-4), is one of the most common human viral pathogens. Primary EBV infection usually begins in the oral cavity. The main target cells are CD21+ B cells and epithelial cells. EBV infection is the cause of infectious mononucleosis (glandular fever) and is associated with various cancers, including Hodgkin's lymphoma, Burkitt's lymphoma, gastric cancer, and nasopharyngeal carcinoma. Upon entering a host cell, EBV may adopt the latent or lytic life cycle. Although EBV predominantly establishes latency in host cells, a few lytically-infected cells could be carcinogenic through the release of growth factors and oncogenic cytokines. Therefore, to better appreciate the risks associated with EBV infection, we need to understand how the virus switches from latency to the lytic cycle in a process termed EBV reactivation. The viral activation can be divided into three phases: Immediate early (IE), Early (E) and Late (L). The viral IE genes BZLF1 and BRLF1 are first transcribed to produce two transactivators, Zta and Rta, which are required for EBV genome replication. These proteins can act synergistically or independently to trigger EBV reactivation. Rta may regulate transcription via three mechanisms of action. Rta may autonomously activate the expression of a certain set of genes, such as BMLF1 which encodes a mRNA export factor. Moreover, Rta and Zta may act in synergy to activate some lytic cycle genes, such as BMRF1. Furthermore, Rta may interact with specificity protein (Sp1) through an intermediary protein, MCAF1, to form a regulatory complex on Sp1-binding sites. Although both Rta and Zta act as transcriptional activators, they exhibit distinct functional and DNA-binding properties. So far, only Zta is biochemically and struturally characterized, much less regarding Rta is known. Because lytic-induction therapy in tumor cells are being regarded as a potential therapy for EBV-positive tumors, and the Rta-mediated reactivation of EBV is the main cause of viral transmission, it would be helpful to understand the function of Rta in greater detail. We aim to determine the crystal structures of Rta, either alone or in complex with the EBV gene promoter element to elucidate its function. Toward this goal, we have successfully expressed 6x His-tagged Rta in BL21-star cells, and the recombinant protein may be purified by a combination of Nickel, heparin and gel filtration column. Purified Rta is homogenous and exists as a homodimer, corresponding to the expected configuration. Electrophoresis mobility shift assay (EMSA) indicates purified Rta may associate with an DNA duplex containing the BMLF1-RRE sequence. Crystallization trials for the Rta dimer and Rta-DNA complex are currently underway. A preliminary crystallization condition for Rta-DNA complex has been identified. This condition will be systematically optimized to produce crystals of higher quality for subsequent crystallographic analysis.