EB病毒單股DNA結合蛋白BALF2的功能區域與其交互作用蛋白質之探討

Abstract

EB 病毒 (Epstein-Barr virus) 是感染多數人的病毒，可能導致感染性單核球增多症或是沒有明顯症狀。EB 病毒第一次感染細胞後，EB 病毒的線性 DNA 會進入細胞核中並形成環狀 DNA，以潛伏期的形式隨著宿主的細胞週期一同複製。當細胞受到刺激時，EB 病毒 BHLF1 RNA 會表現並幫助穩定解螺旋的OriLyt DNA 。此時，溶裂期 DNA 複製蛋白起始點結合因子 Zta、DNA 聚合酶BALF5、聚合酶輔助因子 BMRF1 及病毒單股 DNA 結合蛋白 BALF2 則會結合到 DNA 上，進行溶裂期 DNA 複製。先前實驗室研究發現 BALF2 a.a. 1100-1128 剔除的突變株使 BALF2 無法進核，推測，BALF2 a.a. 1100-1128 對於 BALF2 進核是非常重要的。所以本論文的第一個部份將利用 BALF2 剃除的病毒株 (p2089-BALF2KO) 及 BALF2 a.a. 1113-1117 由 RRKRR 換成 AAAAA，使 BALF2 無法進核的病毒株 (p2089-BALF2NLS5A bacmid) 探討 BALF2 的功能區域。第二部分根據酵母菌雙雜交的實驗結果得知，BALF2 會與 actin 結合蛋白 zyxin 交互作用。因此推測，BALF2 可能有除了結合單股 DNA 的其他功能來幫助 EB 病毒的複製。想藉由與 BALF2 交互作用的蛋白質探討 BALF2 是否有另外的功能。目前實驗結果可分為兩個部分(I) 為了要探討 BALF2 的功能區域，本論文已建構 p2089-BALF2KO 及 p2089-BALF2NLS5A bacmid 並送入可經由 doxycycline 誘導進入溶裂期的 293TetEZ 細胞中。之後可將不同突變的 BALF2 補回293TetEZ p2089-BALF2KO 及 p2089-BALF2NLS5A細胞中，分析對於 BALF2 進核的重要片段。(II) 利用免疫螢光染色實驗發現，EB 病毒再活化後，可觀察到 zyxin 會聚集到細胞核凹陷處，因此 zyxin 可能參與 EB 病毒的成熟。根據免疫螢光染色實驗發現，BALF2 與 zyxin 有些微共位的訊號，但卻無法利用免疫共沉澱觀察到複合體。在 zyxin 表現減量的 NA 細胞中，病毒蛋白的表現量比控制組多，但細胞中的 EB 病毒 DNA 量卻沒有顯著上升。分泌的病毒顆粒量相較於控制組有顯著上升的現象，因此推測zyxin可能會抑制EB 病毒顆粒的分泌。此外也利用 Flag-BALF2 共同免疫沉澱及質譜分析的方法，探討與 BALF2 交互作用的蛋白質。這些結果皆有助於未來對 BALF2 功能的了解。

Epstein-Barr Virus (EBV) infection may be asymptomatic or with Infectious Mononucleosis (IM) in teenagers. After primary infection of EBV, the linear form of viral DNA is circulized into episomal form and replicates coordinately with the cell cycle of host cells. Lytic replication may be induced by starvation of host cells or various stress signals in the environment. EBV BHLF1 RNA begins to be expressed and stabilizes the unwind region of OriLyt. Core replication proteins, such as the origin binding protein Zta, DNA polymerase BALF5, polymerase associated factor BMRF1 and single-stranded DNA binding protein BALF2 coordinately bind onto viral DNA and initiate lytic replication. EBV ssDNA-binding protein, BALF2, plays an essential role for viral DNA replication. Previous immunofluorescence data shown that deletion of BALF2 a.a. 1100-1128 disrupted its nuclear localization, suggesting 1100-1128 is required for BALF2 nuclear localization. The first part of this study was to define the functional domains of BALF2 by BALF2 knockout virus (p2089-BALF2KO bacmid) and trans-complementation. In addition, a bacmid containing the substitution of BALF2 from 1113RRKRR1117 to 1113AAAAA1117 which is known to disrupt nuclear localization (p2089-BALF2NLS5A bacmid) was used to test its effect on virus replication. The second goal of this study was to explore BALF2 function through identifying BALF2-interacting proteins. BALF2 was found to interact with actin-associated protein Zyxin in yeast two hybrid screening, suggesting there may be additional functions for BALF2 to regulate virus replication. To this end, two sets of experiments were performed. (I) In order to reveal the functional domains of BALF2, p2089-BALF2KO and p2089-BALF2NLS5A bacmid were generated and transfected into 293TetEZ cells that can be induced by doxycycline for lytic viral replication. BALF2 deletion mutants were trans-complemented to define the essential regions for nuclear localization and other functional domains of BALF2. (II) According to the data of immunostaining, BALF2 partially colocalized with zyxin. However, the interaction was not detected in co-immunoprecipitation assay. Knockdown of Zyxin with shRNA in NA cells induced spontaneous lytic gene expression and viral DNA replication, suggesting Zyxin may prevent virus lytic replication. In addition, mass spectrometry was performed with transiently transfected Flag-BALF2 and immunoprecipitation approach to identify possible BALF2 interacting proteins. These results provide potential functions of BALF2 for future studies.