EB病毒單股DNA結合蛋白進核機制及與其交互作用蛋白質之探討

Abstract

BALF2為EB病毒複製必需之蛋白質之一，其主要功能為保護單股DNA並避免單股DNA形成二級結構。本研究目的為探討BALF2之進核機制及其與細胞中蛋白質的交互作用，同時利用BALF2缺失之突變病毒株，觀察BALF2於EB病毒溶裂期複製之功能。首先，在免疫螢光染色與細胞核質分離萃取觀察中，發現BALF2之完整結構對於其進核是重要的。經由建立BALF2 a.a.1100-1128與GFP-LacZ的融合蛋白，確認a.a. 1100-1128為BALF2之進核序列，且其中之鹼性胺基酸對於其進核功能是重要的。此外，BALF2之進核亦需要Importin β與Ran-GTP之交互作用。綜合以上觀察，顯示BALF2應是透過典型進核機制進核。另一方面，以免疫螢光染色觀察BALF2與細胞中蛋白質的交互作用，發現在HeLa細胞中表現Flag-BALF2會使高基氏體蛋白GM130表現位置改變。而在帶有EB病毒之NA細胞中轉染Rta質體使EB病毒再活化進入溶裂期時，BALF2會與GM130及披膜蛋白BBLF1聚集於EB病毒造成之細胞核凹陷。推測在EB病毒溶裂期複製時，BALF2可能在此區域與GM130交互作用並參與病毒核殼體組裝。同時觀察BALF2是否會改變actin相聯蛋白zyxin表現位置，然而在HeLa細胞中以免疫螢光染色並未觀察到BALF2表現時影響zyxin之表現位置，共同免疫沉澱亦無觀察到交互作用。最後，嘗試利用BALF2缺失之突變病毒株研究BALF2於EB病毒溶裂期複製之功能。目前已建構BALF2STOP bacmid並將其轉染至293TetZ細胞中，挑選穩定細胞株後可進一步研究BALF2不同區域刪除突變對於EB病毒溶裂期複製之影響。

EBV-encoded ssDNA-binding protein, BALF2, is one of the essential viral replication proteins. The functions of ssDNA-binding proteins are to protect ssDNA and prevent ssDNA from forming secondary structure. The specific aims of this study are to identify the nuclear targeting mechanism, to explore possible biological functions of BALF2 and to utilize BALF2STOP bacmid for studing the function of BALF2 in EBV lytic replication. First, the nuclear targeting of EBV BALF2 was characterized. Immunofluorescence and subcellular fractionation observation showed that the intact BALF2 conformation is important for its nuclear localization. By the fusion of GFP-LacZ, a.a. 1100-1128 of BALF2 were identified as the nuclear targeting signal. Residues substitution of positive charged amino acids within a.a.1100-1128 (NLS5A) abolished BALF2 nuclear targeting. The interaction of importin-β and Ran-GTP is also important for BALF2 nuclear targeting. To explore possible biological functions of BALF2, immunofluorescence assay was used to observe the pattern of BALF2 and the cellular proteins that were found to interact with BALF2 by yeast-two hybrid screening. We found BALF2 altered the expression pattern of GM130, a cis-golgi matrix protein, and partially co-localized with GM130 in the presence of viral proteins, suggesting BALF2 may be involved in viral maturation through interact with GM130. On the other hand, BALF2 didn’t alter the expression of the actin-associated proteins zyxin. To further study the function of BALF2 in EBV lytic replication, a BALF2-stop bacmid (p2089BALF2STOP) was generated and transfected to 293TetEZ cells. After selection, the stable cell pool clones can be used to study the effect of different functional domains of BALF2.