EB病毒尿嘧啶醣苷酶BKRF3在溶裂期核酸複製區之功能

Abstract

尿嘧啶醣苷酶(uracil DNA glycosylase)是一群能夠移除DNA上尿嘧啶(Uracil)，參與在鹼基切除修復(BER)的酵素。BKRF3為EB病毒在溶裂期早期轉錄轉譯出來的尿嘧啶醣苷酶。先前的研究發現BKRF3以及細胞中的UDG (UNG2)會參與在EB病毒溶裂期複製當中。而觀察此時蛋白質表現發現，隨著在上皮細胞溶裂期複製的進行，BKRF3表現量會逐漸上升，UNG2表現量會逐漸下降，推測在此時BKRF3可以補足UNG2下降所喪失的尿嘧啶醣苷酶活性，以維持病毒複製的效率。此外，外送BKRF3後提引細胞進入溶裂期複製，可觀察到其從細胞質轉移到細胞核的現象。因此，本篇論文的目的是要觀察BKRF3與其他病毒蛋白交互作用後，對於其進入到細胞核，以及對本身酵素活性，最後對病毒DNA複製的影響。第一部分利用免疫共沉澱法觀察到BKRF3與病毒核糖核苷酸還原酶(RR)交互作用可能十分微弱，因此未來還需進一步研究其他溶裂期早期病毒蛋白是否可以協助BKRF3入核，參與在病毒核酸複製區中。第二部分利用尿嘧啶醣苷酶酵素活性試驗發現溶裂期時，細胞內的尿嘧啶醣苷酶酵素活性可以維持在某一定程度，考慮到BKRF3純化出來的酵素活性低，此實驗結果暗示BKRF3的活性在溶裂期時可受刺激而提高，利用IP-UDG assay也證明BKRF3的酵素活性的確有上升的現象。另外，構建BKRF3缺失之突變病毒株來觀察BKRF3在溶裂期複製時所扮演的角色。目前已將此突變株轉染至293TetER細胞中，並已獲得兩株細胞株。未來可利用QPCR實驗進一步證明BKRF3參與在EB病毒溶裂期核酸複製區中。

Uracil DNA glycosylase (UDG) is an enzyme that participates in the DNA base excision repair (BER). BKRF3 is a viral UDG encoded at the early phase of EBV lytic stage. Previous data have shown both BKRF3 and the cellular major UDG (UNG2) are involved in EBV lytic replication. Protein expression kinetics revealed that BKRF3 is upregulated, but UNG2 is downregulated along the progression of lytic cycle in epithelium cells, suggesting BKRF3 may compensate the UNG2 activity during virus lytic cycle to sustain the viral DNA replication efficiency. Besides, exogenous expression of BKRF3 can be translocated from cytoplasm to nucleus after lytic cycle induction. In this study we aimed to investigate the interaction between BKRF3 and other viral proteins that required for its nuclear localization, enzyme activity enhancement and viral DNA replication. The first part of experiments demonstrated that the interaction between BKRF3 and viral ribonucleotide reductase (RR) may be weak in co-IP assay. Therefore, other early viral proteins will be examined for possible interactions. In the second part, UDG activity assay demonstrated that total UDG activity was maintained at a particular level throughout EBV replication. Because the activity of the purified BKRF3 is low, the data suggest the UDG activity may be stimulated during lytic replication. IP-UDG assay revealed that the UDG activity was elevated during lytic cycle. Additionally, BKRF3-null EBV bacmid was generated to investigate the role of BKRF3 in viral replication. The mutant bacmid had been transfected into 293TetER cell line and two stable cell lines had been selected. Further experiments will be performed to prove the role of BKRF3 in viral replication compartment.