EB病毒BSLF1蛋白質的功能性分析

Functional Analysis of BSLF1 of Epstein-Barr Virus

林庭羽(Ting-Yu Lin)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/生化科技學系/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10847

摘要

Epstein-Barr virus (EB病毒) 屬於人類疱疹病毒科，其生活史包含了潛伏期及溶裂期。EB病毒在溶裂期會依序表現極早期、早期及晚期基因，而藉由這些蛋白質的共同表現才能產生具有感染性的病毒顆粒。極早期蛋白質Rta與Zta為兩個病毒的轉錄因子，能促進複製蛋白質的表現、協助病毒基因體的複製及生成組裝病毒顆粒所需要的結構性蛋白質。先前研究指出Zta能結合在溶裂期複製起始點 (oriLyt) 上，並協助聚集複製蛋白質以組裝成完整的複製複合體。除了Zta之外，還需要其他六種複製蛋白質的共同參與才能順利開啟複製，而本研究主要以病毒的引子酶 (primase) BSLF1作為主要研究目標。首先，藉由共免疫沉澱分析及GST pull-down證明了Rta與BSLF1能直接結合，且BSLF1會與Rta蛋白質N端190個胺基酸片段及N190-415片段結合。接著，利用293T細胞進行免疫螢光染色分析，發現Rta會與BSLF1及BBLF2/3共同位在細胞核內，而Rta各別會與BSLF1或BBLF2/3主要分布在細胞質中。此外，觀察以TPA及sodium butyrate (SB/TPA) 誘導病毒進入溶裂期36小時的P3HR1細胞，發現Rta會與BSLF1及BBLF2/3呈點狀分布於細胞核內，顯示病毒在進行複製時Rta會位在複製複合體中。而在先前的研究中，預測了BSLF1可能具有去泛素化酵素的活性，本研究也利用體外去泛素化酵素活性分析證明BSLF1具有截切泛素的能力，而在細胞中過量表現BSLF1時能移除Rta上的泛素化修飾。此外，不論在P3HR1或293T細胞中過量表現BSLF1都能增加Rta在細胞中的含量，顯示BSLF1移除Rta上的泛素化修飾可能有助於增加Rta的穩定性。最後，本研究發現BSLF1會降低Rta活化BMRF1及BMLF1啟動子的能力，推測可能與調節病毒溶裂期基因的表現量相關。綜合以上結果，本研究發現BSLF1能夠移除Rta上的泛素化修飾並進而調節Rta的功能。此外，Rta也被發現會與BSLF1及BBLF2/3共同分布在細胞核內，顯示Rta可能參與在病毒的溶裂複製上。

關鍵字

EB病毒 ； Rta ； BSLF1 ； BBLF2/3 ； 去泛素化修飾

並列摘要

Epstein-Barr virus (EBV) is a human herpesvirus with two distinct life cycles, latent and lytic. During the lytic cycle, EBV expesses the immediate-early, early and late genes in order to produce infectious virus particles. Rta and Zta are two transcription factors expressed during the immediate-early stage, and both of them are required to trigger sequential events including expression of replication proteins, amplification of viral genome and synthesis of structural proteins. Previous study showed that Zta acts as an oriLyt-binding protein, facilitating the assembly of viral replication machinary.Besides Zta, there are other six proteins involved in lytic replication. In this study, we aim to explore the functions of BSLF1, which is a primase. GST pull-down and coimmunoprecipitation studies reveal that Rta interacts with BSLF1, and the interaction involves the N-terminal and the middle region in Rta. Moreover, indirect immunofluorescence analysis indicates that Rta, BSLF1 and BBLF2/3, a primase-associated protein, colocalize in the nucleus, while Rta and BSLF1 or BBLF2/3 colocalize mainly in the cytoplasm in 293T cells. Furthermore, Rta, BSLF1 and BBLF2/3 colocalize in the nucleus at 36 hr after lytic induction in P3HR1 cells, indicating that Rta may be involved in the replication complex. In addition, BSLF1 has been shown to function as a deubiquitinase (DUB). Overexpression of BSLF1 decreases the ubiquitination of Rta and increases the stability of Rta. BSLF1 also reduces the transactivation activity of Rta which may modulate the expression of lytic genes. Taken together, this study demonstrates that BSLF1 is capable of deubiquitinating Rta and modulating the functions of Rta. BSLF1 also interacts with BBLF2/3 and Rta, suggesting that Rta is involved in lytic replication.

並列關鍵字

Epstein-Barr virus ； Rta ； BSLF1 ； BBLF2/3 ； deubiquitinase (DUB)

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