泛素化修飾與EB病毒Rta及Zta的協同作用

Abstract

Epstein-Barr virus (EB病毒) 具有潛伏期及溶裂期兩個生活史。當病毒感染B細胞或上皮細胞後，一般會以潛伏的狀態存在，但其必須進入溶裂期才能產生病毒顆粒而感染其他細胞。在溶裂極早期，EB病毒會表現Rta與Zta兩個轉錄因子，能以協同合作的方式大量活化病毒的早期基因。本實驗室先前的研究證明了MCAF1參與Rta與Zta的協同作用，但是否尚有其他細胞蛋白質參與調控目前仍不清楚。RNF4是一個具有RING區域的泛素E3連接酶，能催化受SUMO修飾的PML進行泛素化修飾。本研究的第一部份探討RNF4如何調控Rta的蛋白質含量。首先，本研究發現以26S proteasome抑制劑MG132處理細胞後，會使受SUMO修飾的Rta更為穩定並促進EA-D的表現。同時，GST pull-down與免疫沈澱分析的結果指出Rta能與RNF4相互結合。在細胞外及細胞內泛素化修飾分析的結果發現，RNF4能將受SUMO-2修飾的Rta進行泛素化修飾，且此功能必須透過RNF4蛋白質上的SUMO-interaction motifs (SIMs) 區域。此外，若將Rta受SUMO-2/3修飾的lysine突變，RNF4對其泛素化修飾的程度明顯降低，顯示RNF4為Rta的SUMO-targeted ubiquitin E3 ligase (STUbL)。當RNF4的表現受到抑制時，Rta及EA-D的表現亦受到影響，因而降低了EB病毒的複製及病毒顆粒的產生。另外，RNF4亦能與Zta結合，而Zta可透過拮抗RNF4之作用使Rta的泛素化修飾減少，並增加Rta的穩定性。本研究證明了SUMO-targeted ubiquitin E3 ligase參與調控Rta的泛素化修飾，進而影響EB病毒溶裂期的進行。另一方面，RanBPM能與許多蛋白質結合，因此被視為一個多功能的蛋白質而影響細胞的生理活性。先前的研究指出RanBPM與Rta能互相結合。因此本研究的第二部份主要探討RanBPM在Rta與Zta協同作用中所扮演的角色。實驗結果顯示，Zta能與RanBPM結合，結合位置位在RanBPM的SPRY區域與Zta的C端。Rta與 Zta能透過RanBPM的連接而形成Zta-RanBPM-Rta複合體，並能結合在在BHLF1，BRLF1和BHRF1啟動子上的ZRE序列。當外送RanBPM的shRNA降低其細胞中的含量時，Rta與Zta所引發的協同效應明顯降低，且過量表現RanBPM亦能促進EB病毒溶裂期的進行。另外，Rta與 Zta能透過去泛素化酵素，USP11，將RanBPM的泛素化修飾去除而增加其穩定性，進而穩定Rta-RanBPM-Zta複合體。綜合以上結果，本研究從後轉譯修飾的角度解釋Rta與Zta協同作用的機制，並對 EB病毒溶裂期的發展提供更進一步的了解。

Epstein-Barr virus (EBV) has two life cycles. After infecting B lymphocyte or epithelial cells, the virus typically remains latent. However, EBV must enter a lytic cycle to proliferate and produce infectious particles. During the immediate-early stage of the lytic cycle, EBV encodes two transcription factors, Rta and Zta, to activate synergistically the transcription of the genes required for viral lytic development. Our previous study demonstrated that MCAF1 is involved in the synergistic activation by Rta and Zta. However, whether another cellular factors regulate the synergy remains unclear. RNF4 is a RING-domain-containing ubiquitin E3 ligase that targets sumoylated PML for ubiquitination. In the first part of the dissertation, this study investigates how the Rta protein level is regulated by RNF4. This work demonstrates that treating P3HR1 cells with a proteasome inhibitor, MG132, causes the accumulation of SUMO-Rta and promotes the expression of EA-D. GST pull-down and coimmunoprecipitation assays reveal that RNF4 interacts with Rta. RNF4 also targets SUMO-2-conjugated Rta and promotes its ubiquitination in vitro. Additionally, SUMO-interaction motifs (SIMs) in RNF4 are important to the ubiquitination of Rta because the RNF4 mutant with a mutation at the motifs eliminates ubiquitination. The mutation of four lysine residues on Rta that abrogated SUMO-2/3 conjugation to Rta also decreases the enhancement of the ubiquitination of Rta by RNF4. This finding demonstrates that RNF4 is a SUMO-targeted ubiquitin E3 ligase (STUbL) of Rta. Moreover, knockdown of RNF4 enhances the expression of Rta and EA-D, subsequently promoting EBV lytic replication and virions production. Furthermore, Zta interacts with RNF4 to antagonize the ubiquitination of Rta by RNF4, thus stabilizing Rta. Results of this study significantly contribute to efforts to elucidate a SUMO-targeted ubiquitin E3 ligase that regulates Rta ubiquitination to influence the lytic development of EBV. On the other hand, RanBPM is concerned a multifunctional protein that interacts with a broad spectrum of proteins to influence the cellular physiological functions. An earlier study indicated that RanBPM interacts with Rta. In the second part, this study investigates whether RanBPM is involved in the synergy of Rta and Zta. This work demonstrates that RanBPM binds to Zta in vitro and in vivo. The interaction appears to involve the SPRY domain in RanBPM and the C-terminal domain in Zta. Furthermore, this study indicates that Zta and Rta form a complex via an intermediary protein, RanBPM, in vitro. The Zta-RanBPM-Rta complex binds to the ZREs in the BHLF1 and BHRF1 promoters. Additionally, introducing the shRNA of RanBPM into cells reduces the synergistic activation of lytic promoters by Rta and Zta. Overexpression of RanBPM also enhances the EBV lytic cycle. Moreover, USP11 associates with the Rta-Zta complex to prevent the ubiquitination of RanBPM, thus stabilizing the synergy complex. Taken together, this study provides the insight from the view of ubiquitination to understand the mechanism how Rta and Zta synergistically activate its lytic genes, thus favoring lytic progression.