Characterization of Epstein-Barr virus BALF3 gene product

EB病毒的開放譯讀架BALF3，經胺基酸序列比對發現與疱疹單純病毒 (HSV)的UL28基因屬於同一個具高度保守性的基因家族，此基因在疱疹病毒科可能的功能為末端酶 (terminase)，與病毒的核蛋白質殼（nucleocapsid）成熟有關。然而過去從未有研究報導過BALF3在EB病毒進入溶裂期時是否表現。我們將含有EB病毒基因體的Akata細胞以抗IgG抗體(induction)後，利用一特定anti-sense RNA做為探針進行北方墨點法分析，可在提引後6小時偵測到一大約6.0 kb包含BALF3的RNA。利用特定的引子做反轉錄－聚合酵素鏈鎖反應（RT-PCR）亦可偵測到包含BALF3的RNA表現，推測此6.0 kb RNA是可能由BALF2啟動子所驅動，表現涵蓋BALF2及BALF3兩個基因的轉錄產物。將BALF3從EB病毒基因體選殖出來，並且在大腸桿菌裡表現帶有六個His標籤的BALF3 (EBV B95-8 strain：158851-160906(L))融合蛋白質。純化後的融合蛋白用以製作辨認BALF3蛋白質的老鼠血清。利用此血清以免疫墨點法在轉染BALF3表現質體的293T 細胞中，可以偵測到Flag-BALF3蛋白質。而在以TPA/SB提引的NA細胞及以goat anti-human IgG提引的Akata細胞中亦可偵測到BALF3蛋白的表現。進一步分析BALF3和其它EB病毒蛋白質進入溶裂期時表現量的變化，我們發現BALF3為溶裂期早期表現之病毒蛋白。另外利用老鼠血清以免疫螢光染色法偵測BALF3在短暫轉染的293T細胞中的表現位置，結果發現在沒有其他EB病毒蛋白質存在的情況下，BALF3主要位於細胞質。最後，以純化的His-BALF3融合蛋白作為抗原，分析血清中抗BALF3的IgA及IgG抗體，發現在16個鼻咽癌患者中4個有辨識BALF3的IgA (25%)，10個有辨識BALF3的IgG (62%)；而在9個健康捐贈者中則未測到抗體力價，顯示病毒在人體內複製時，確實有BALF3蛋白質的表現。至於BALF3蛋白質在病毒複製的細胞中所表現的位置及其生物功能，或是其抗體力價與疾病相關性則仍需進一步的研究。Epstein-Barr virus (EBV) BALF3 is a homologue of the herpes simplex virus (HSV) UL28 gene, which encodes a putative terminase component conserved among herpesviruses. Terminase plays a role in recognition of pac sequences within the DNA termini of herpevirus, and in cleavage of the concatemeric DNA. The cleavage is required for correct package of viral genome and maturation of viral nucleocapsid. However, the expression of BALF3 has not been demonstrated previously. An anti-sense RNA probe detected a specific 6.0 kb transcript in Akata (EBV+) 6 h post-induction. A transcript containing BALF3 was also detected by RT-PCR using specific RT primers. These results suggest BALF3 containing transcript is expressed in EBV replicating cells, and the transcript is probably driven by BALF2 promoter. To characterize the expression of BGLF3 protein in EBV replicating cells, we then generated BALF3 specific sera. His-tag fused BALF3 protein was expressed in Escherichia coli and purified with nickel beads. The purified protein was used to generate mouse and rabbit specific antisera. Using BALF3 specific sera, we detected the expression of Flag-BALF3 fusion protein in transfectly transfected-293T cells in immunoblotting; and the fusion protein was observed in the cytoplasm of transfected cells in immunoflourescence assay. BALF3 was also detected with specific serum in EBV positive Akata and NA cells, and the detected molecular weight of BALF3 is about 75-kDa. Simultaneous detection of expression kinetics of BALF3 and other viral proteins revealed that BALF3 expresses as an early protein. Finally, we tested whether BALF3 antibody is present in the sera of nasopharyngeal carcinoma (NPC) patients using bacterially purified recombinant BALF3 protein as antigen. As a result anti-BALF3 IgA was detected in 4 of 16 (25%) and IgG was detected in 10 of 16 (62%) patients, but not detected in all 9 healthy donors. This observation further supports the in vivo expression of BALF3 in EBV infected individuals. Further studies are required to identify the sub-cellular localization and biological functions of BALF3 within EBV replicating cells.中文摘要 I 英文摘要 II 前言 1 研究目標 12 材料與方法 13 結果 22 討論 28 圖表 34 參考文獻 5