Ku80與EB病毒Zta蛋白質的功能研究

Ku80 and the Functions of Zta of Epstein-Barr Virus

陳建彰(Chien-Chang Chen)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.00793

摘要

EB病毒 (Epstein-Barr virus；EBV)屬於一種人類疱疹病毒，當病毒進入溶裂期會表現出極早期基因BZLF1並轉譯出Zta蛋白質，Zta為一個多功能蛋白質，主要負責調控基因轉錄及將DNA複製相關的蛋白質聚集到複製起始點。為了探討Zta在細胞中是否與其它細胞內蛋白質結合，對EB病毒溶裂期機制造成影響，所以本研究利用His pull-down assay吸附細胞蛋白質並以MALDI-TOF進行蛋白質定序發現人類的ATP-dependent DNA helicase II Ku80會與Zta結合，接著進一步以共同免疫沉澱分析和GST-pull down證實了Zta會與Ku80在細胞內及細胞外結合並一起結合到oriLyt上，並且觀察到Zta以C端區域與Ku80的N端區域結合，也在Ku80缺陷的P3HR1細胞中觀察到EB病毒溶裂期DNA複製的下降，Ku80也會與Zta和EA-D在細胞內結合。此外，利用冷光分析發現Ku80會增加Zta的轉錄活性。綜合以上，Zta與Ku80結合後會進一步調控EB病毒溶裂期複製。

關鍵字

人類疱疹病毒第四型 ； Zta ； Ku80 ； 溶裂期 ； DNA複製

並列摘要

Epstein-Barr virus (EBV) expresses immediate-early transcription factor Zta during the lytic cycle. Zta is a multifunctional protein that regulates gene transcription and recruits a replication complex at the lytic viral origin of replication. To identify cellular proteins that associate with Zta, this study performed His-Zta pull down analysis to precipitate cellular proteins and identified by MALDI-TOF a human ATP-dependent DNA helicase II Ku80 that interacts with Zta. Furthermore, Zta interacts with Ku80 in vivo and in vitro, forming a complex that binds to ZREs at the oriLyt. Furthermore, depleting Ku80 reduces EBV lytic DNA replication, showing the involvement of Ku80 in EBV lytic development. This study shows that the C-terminal domain of Zta interacts with the N- and M-domain of Ku80. Ku80 and Zta also interact with BMRF1 polymerase processivity factor, suggesting that Ku80 facilitates viral lytic DNA replication . In addition, Ku80 enhances the transcriptional activity of Zta. Taken together, this study found Ku80 involves in EBV lytic replication through an interaction with Zta.

並列關鍵字

Epstein-barr virus ； Zta ； Ku80 ； Lytic cycle ； DNA replication

參考文獻

Adams A (1987) Replication of latent Epstein-Barr virus genomes in Raji cells. J Virol 61(5): 1743-1746

Aiyar A, Tyree C, Sugden B (1998) The plasmid replicon of EBV consists of multiple cis-acting elements that facilitate DNA synthesis by the cell and a viral maintenance element. EMBO J 17(21): 6394-6403

Andersson-Anvret M, Forsby N, Klein G, Henle W, Biorklund A (1979) Relationship between the Epstein-Barr virus genome and nasopharyngeal carcinoma in Caucasian patients. Int J Cancer 23(6): 762-767

Baumann M, Feederle R, Kremmer E, Hammerschmidt W (1999) Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt. EMBO J 18(21): 6095-6105

Bryant H, Farrell PJ (2002) Signal Transduction and Transcription Factor Modification during Reactivation of Epstein-Barr Virus from Latency. J Virol 76(20): 10290-10298