I、Characterization of nuclear localization regulation of Epstein-Barr virus BGLF4 kinase，II、Establishment of an expression system of BGLF4 kinase in recombinant baculovirus

BGLF4是在EB病毒中唯一已知的Ser/Thr蛋白質激酶，它可以磷酸化病毒DNA聚合酵素輔助因子(BMRF1)、病毒細胞核抗原(EBNA2, EBNA-LP)或細胞轉譯延長因子(EF-1δ)等受質。BGLF4分布在轉染或帶有病毒複製細胞之細胞核中。根據先前觀察中發現，去除掉BGLF4羧基端(Carboxy terminus, C)會導致其喪失核定位。同時於羧基端胺基酸386 RSLKKRFK 393處為一富有鹼性胺基酸區域，疑似存有潛在性核定位訊號(nuclear localization signal, NLS)。因此本篇想要探討BGLF4上針對核定位與其激酶活性之功能性區域。去除胺基酸386-393後，BGLF4喪失其核定位與激酶活性。然而將其區域之精胺酸、離胺酸(Arg, Lys)突變為丙胺酸(Ala)，發現可能是影響BGLF4之結構而非核定位訊號。為了更進一步辨認端上之兩段可能之alpha-helix對於BGLF4核定位與激酶活性之貢獻，建構出d(378-389)與d(410-419)兩蛋白，發現d(378-389)表現於細胞核中，而d(410-419)喪失其核定位及其活性。最後想確認BGLF4核定位是否需要其激酶活性，發現BGLF4 K102I (Catalytic domain上Lys突變為Ile)與D195A (catalysis domain上Asp突變為Ala)皆喪失活性但卻分布於細胞核中，推論BGLF4之核定位是不需仰賴其激酶活性。總而言之，BGLF4上並無帶有傳統性之核定位訊號，同時羧基端上可能之alpha-helix對於其核定位與激酶活性扮演重要角色。 除此之外，為了建立更足夠蛋白質進行激酶活性試驗，我們利用baculovirus表現系統建立重組BGLF4蛋白質激酶。此重組蛋白經由免疫沉澱激酶活性試驗觀察到重組BGLF4蛋白具有激酶之活性。未來可利用此系統純化重組蛋白，針對BGLF4潛在性受質，作更進一步地試驗。BGLF4 kinase is the only identified ser/thr protein kinase of Epstein-Barr virus (EBV). It can phosphorylate several viral substrates such as viral DNA polymerase processivity factor BMRF1, nuclear antigen EBNA-2 and EBNA-LP, and also cellular translation elongation factor 1 delta (EF1δ). BGLF4 expresses predominantly in the nucleus of transiently transfected and virus replicating cells. Based on the observation that the deletion of the carboxyl end of BGLF4 abolished its nucleus localization, the basic amino acid rich sequence between 386RSLKKRFK393 was predicted as the nucleus localization signal (NLS) of BGLF4. In this study we aimed to study the functional domains contributing to the kinase activity and nucleus localization of BGLF4. The kinase activity of BGLF4 was examined by in vitro kinase assay and co-expression of BMRF1 and BGLF4 in HeLa cells. Deletion mutant analysis revealed that a.a. 386-393 are required for proper nucleus translocation and kinase activity. However, substitution of arginine (R) or lysine (L) with alanine (A) within this region indicated that probably the structure rather than the positive charges of this region contribute to nuclear translocation of BGLF4. To identify possible contribution of two predicted alpha-helix regions within the carboxyl terminus of BGLF4, d(378-389) and d(410-419) were generated. We found that d(410-419) lost kinase activity as demonstrated by in vitro kinase assay and by co-expression of BMRF1 and BGLF4 in HeLa cells . Notably, d(378-389) but not d(410-419) maintains its nucleus distribution. To examine whether kinase activity is required for nuclear localization, K102I (with a mutation at catalytic lysine at a.a. 102) and D195A (with a mutation at catalysis domain) were observed for their predominantly nucleus localization, suggesting nucleus translocation of BGLF4 is not regulated by its own kinase activity. Taken together, results in this study suggest that BGLF4 does not contain a classical NLS and the putative helix regions within carboxyl terminus are important regulatory domains for kinase activity and nucleus localization. Additionally, in order to obtain adequate amounts of protein for in vitro kinase assay, a recombinant baculovirus BGLF4 expression system was established. The recombinant BGLF4 protein activity was demonstrated by immunoprecipitation kinase assay, suggesting this system can be applied to further purification and screening candidate BGLF4 substrates in the future.I、中文摘要------2 II、英文摘要-----3 III、前言--------4 IV、研究目標-----13 V、材料與實驗方法--14 VI、實驗結果-------23 VII、討論----------29 VIII、圖表---------34 IX、參考文獻-------5