EB病毒BGLF4蛋白激酶特性及其抑制干擾素調節因子3
訊息傳遞功能之研究

EB病毒感染全世界超過百分之九十的人口，而且和一些惡性腫瘤例如淋巴癌、鼻咽癌、胃癌以及移植後淋巴癌有高度相關性。為了更加了解這些疾病和病毒致病因子之間的關係，在本研究中，探討EB病毒所合成出以辨識脯胺酸絲胺酸/酥胺酸(Ser/Thr Pro)的BGLF4蛋白激酶，在病毒致病因子和宿主細胞之間的交互作用及其生物意義。為了探討BGLF4的可能功能，首先利用BGLF4的專一性單株抗體，偵測BGLF4在EB病毒複製的細胞中主要位在核內，同時也被包裹在病毒顆粒中。經由酵母雙雜交系統的篩選，發現干擾素調控因子3 (IRF3)的剪接變異體可以和BGLF4蛋白交互作用，並經短暫性轉染共同沉澱法和試管內GST-融合蛋白沉澱方法確定其交互作用。在利用報導質體分析中，BGLF4有效抑制poly(I:C)所刺激的干擾素β啟動子和IRF3反應區域的活性。而且BGLF4有效的抑制內生性干擾素βmRNA的表現以及STAT1 (Signal Transducers and Activators of Transcription protein 1)在第701個胺基酸酪氨酸的位置的磷酸化。進一步探討可能的機轉，BGLF4不影響poly(I:C)所引起IRF3形成雙聚體，轉移至核內，或是與CBP (cAMP Response Element Binding Protein)結合的能力。利用染色質免疫沉澱分析法，發現BGLF4會減少被活化的IRF3結合在帶有IRF3反應區域的之干擾素β啟動子上。BGLF4可以在試管內磷酸化GST-IRF3。利用點突變的分析發現絲胺酸339-脯胺酸340的磷酸化所引發Pin1的調控方式並不涉及BGLF4對IRF3的抑制作用；而是位於IRF3 DNA結合區域和干擾素調控因子結合區域中間的3個SP序列，包括Ser123、Ser173及Thr180的磷酸化可以加成性地反應BGLF4抑制IRF3的轉活化功能。同時發現在被活化進入溶裂期的EB病毒陽性NA細胞中，IRF3 也被活化。若送入siRNA抑制BGLF4的表現量，則帶有IRF3反應區域的報導質體活性會被提高。本研究的結果發現疱疹病毒的蛋白激酶可以透過一種新的機制抑制細胞的干擾素活化，以利病毒複製。

關鍵字

蛋白激酶干擾素調節因子3

並列摘要

Epstein-Barr virus (EBV) infects more than 90% worldwide population, and is highly associated with many malignancies such as lymphoma, nasopharyngeal carcinoma (NPC), gastric carcinoma, and post-transplantation lymphoma diseases. In this study, EBV encoded proline-directed ser/thr protein kinase BGLF4 was studied for the interplay between virus virulent factors and the host factors and the biological significance of the interaction. To search for the possible functions of BGLF4, specific monoclonal antibodies (MoAbs) were generated to demonstrate that BGLF4 mainly localized in the nucleus of the EBV positive epithelial cells, and it is a virion-associated protein kinase. Moreover, through a yeast two-hybrid screening approach, a splicing variant of interferon regulatory factor 3 (IRF3) was found to interact with BGLF4 protein. This interaction was further defined by co-immunoprecipitation in transfected cells and GST pull-down in vitro. Using reporter assays, BGLF4 effectively suppressed the poly(I:C)-stimulated IFNβ promoter and IRF3 responsive element activities. Moreover, BGLF4 repressed poly(I:C)-stimulated expression of endogenous IFNβ mRNA and phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 did not affect dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C)treatment. Notably, BGLF4 reduced the amount of active IRF3 recruited to the IRF3 responsive element (IRE) containing the IFNβ promoter region in a ChIP assay. BGLF4 phosphorylated GST-IRF3 in vitro, but Ser339-Pro340 phosphorylationdependent, Pin1-mediated down regulation is not responsible for the repression. Most importantly, three proline-dependent phosphorylation sites at Ser123, Ser173 and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contributed additively to BGLF4 mediated repression of IRF3(5D) transactivation activity. IRF3 signaling was activated in reactivated EBV positive NA cells, and knockdown of BGLF4 further stimulated IRF3 responsive reporter activity. The data presented here thus provide a novel mechanism by which herpes viral protein kinases suppress host innate immune responses and facilitate virus replication.

並列關鍵字

BGLF4 kinase ； interferon regulatory factor 3

參考文獻

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EB病毒BGLF4蛋白激酶特性及其抑制干擾素調節因子3 訊息傳遞功能之研究

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