探討EB病毒BFRF1蛋白質功能區域以及入核機制之研究

Abstract

中文摘要 當EB病毒在細胞核內完成溶裂期複製(lytic replication)與衣殼化(encapsidation) 之後，會透過出核複合體(Nuclear egress complex) BFRF1/BFLF2的幫助運送到細胞質中，完成後續病毒顆粒的成熟以及釋出步驟。在先前實驗室的研究中發現BFRF1可以透過吸引Alix的方式，藉由ESCRT模組(Endosomal sorting complex required for transport machinery)幫助病毒的出核。在單獨表現BFRF1時，BFRF1就能夠由核膜上產生囊泡(vesicles)釋放到細胞質中。先前實驗室研究發現BFRF1可以透過兩個區域與Alix進行交互作用，分別是Late domain 1 (LD1)區域與Alix的Bro區域可以進行交互作用，另外也可以透過EBV specific region (ESR)區域與Alix的proline rich region (PRR)進行交互作用。在本篇研究中發現ESRBFRF1-PRRAlix之間的交互作用會因為核酸酶的作用而降低，顯示ESR-PRR之間的交互作用可能有核酸的參與。另外在共軛焦顯微鏡觀察不同變異的BFRF1分子在細胞中的分布位置時發現ESR區域的刪除會使得BFRF1分子分散在細胞質中。更進一步使用細胞離拆 (cell fractionation) 分離細胞核與細胞質, 發現缺少ESR區域會使得BFRF1分布在細胞質中，因此我們推測ESR區域對BFRF1入核是重要的。利用內輸蛋白-β (importin β)的抑制劑Importazole (IPZ) 處理細胞之後發現BFRF1仍能夠與內膜蛋白emerin產生共位的現象，這個結果推測BFRF1的入核過程不需要內輸蛋白-β的幫助。為了探討BFRF1入核的可能途徑，我們更發現GFP-Alix在BFRF1共同表現的情況下，會分佈至細胞核中。我們推測BFRF1的入核機制可能需要Alix以及ESCRT模組的幫助。最後我們發現在帶有EB病毒的上皮細胞NA中表現CHMP1以及CHMP2等ESCRT-III分子的顯性抑制突變(dominant negative mutants)會導致釋出的病毒量減少。未來仍需進一步釐清不同ESCRT-III分子在EB病毒的成熟過程中所扮演的角色。

Abstract After Epstein-Barr virus (EBV) lytic DNA replication and encapsidation in the nucleus, nucleocapsids are transported into the cytoplasm through a nuclear egress complex (NEC) BFRF1/BFLF2 mediated mechanism for virion maturation and release. Previously, we demonstrated that BFRF1 is able to interact with Alix, which is an adaptor protein of endosomal sorting complex required for transport (ESCRT), to recruit the ESCRT machinery for viral nuclear egress. By expression alone, BFRF1 is able to induce nuclear envelope-derived cytoplasmic vesicles. Two domains of BFRF1 were mapped to interact with Alix, namely late domain 1 (LD1) interacts with BCK1-like resistance to osmotic shock protein 1 (Bro1) domain of Alix and EBV specific region (ESR) interacts with proline rich region (PRR) of Alix. In this study, we demonstrate that the interaction between ESRBFRF1 and PRRAlix decreased after nuclease treatment, suggesting this interaction is mediated by nucleic acids. In confocal microscopy, we found deletion of ESR domain (BFRF1 ESR) caused BFRF1 to diffuse in the cytoplasm. Furthermore, BFRF1 ESR was fractionated into the cytoplasmic fraction, suggesting ESR domain is required for the nuclear targeting of BFRF1. In the presence of importin β inhibitor Importazole (IPZ), BFRF1 still co-localizes with emerin, which is the inner nuclear membrane (INM) marker. It suggests that the nuclear membrane targeting mechanism of BFRF1 is independent of importin β. In addition, we found GFP-Alix is redistributed into the nucleus with BFRF1 co-expression. It suggests that the nuclear targeting pathway of BFRF1 may require Alix and ESCRT machinery. Finally, we showed that expression of dominant negative CHMP1 or CHMP2 in EBV positive epithelial cells NA leads to the decrease of virion secretion. The exact roles of these ESCRT-III components in EBV maturation process need to be further characterized.