EB病毒核膜相關蛋白在病毒出核過程之研究

Abstract

EB病毒(Epstein-Barr virus)順利感染細胞後，進入溶裂期時，病毒DNA會在宿主細胞核中複製，之後被病毒的核殼蛋白(capsid proteins)包裹，組裝成病毒核殼體(nucleocapsid)。先前的研究中發現，EB病毒所轉譯出的兩種核膜相關性蛋白BFRF1以及BFLF2，對於核殼體的出核(nuclear egress)過程是重要的。然而，目前的研究對於這些大型的病毒核殼體如何穿越結構緻密的板蛋白複合體(nuclear lamina)以及內、外核膜，並進入細胞質以利進一步的病毒顆粒成熟及修飾的過程，仍不清楚。本研究中發現在有EB病毒複製的NA細胞中，病毒蛋白BFRF1會使ESCRT (cellular endosomal sorting complex required for transport)機制中的Alix蛋白聚集至細胞核附近，並在核膜及細胞質中的液泡(vesicle)構造上有共位的現象發生。進一步利用單一基因轉染的方式，發現BFRF1單獨表現時，即有能力利用ESCRT機制來形成由核膜衍伸出的液泡構造。除了表現ESCRT機制的決定因子—顯性抑制(dominant negative)形態的Vps4，也在Alix基因抑制的細胞中，發現BFRF1形成液泡結構的能力消失，並且使病毒的核殼體堆積在細胞核中無法出核。結果證實了Alix對於BFRF1液泡結構形成的重要性，並發現多個BFRF1上的結構域和Alix蛋白上的Bro及PRR結構域，對於兩者之間的交互作用是重要的，並證實EB病毒會藉由病毒蛋白BFRF1的幫助，使ESCRT機制中的Alix蛋白聚集到核膜來幫助病毒核殼體的出核。另一方面，藉由阻擋細胞當中自由形式泛素(ubiquitin)的形成，或是表現病毒激酶BGLF4，就能阻止BFRF1形成液泡結構，暗示BFRF1蛋白上磷酸化(phosphorylation)及泛素化(ubiquitination)後修飾之間的交互調控，對於BFRF1所主導的功能是重要的。總而言之，在本研究中發現，EBV的BFRF1蛋白會協同細胞內的ESCRT機制，調節細胞核膜的結構，來幫助EB病毒核殼體能夠順利出核。

After DNA replication and encapsidation, the nucleocapsids of Epstein-Barr virus (EBV) must translocate from the nucleus to the cytoplasm. Viral BFRF1 and BFLF2 are nuclear envelope-associated viral proteins indispensable for the nuclear egress of nucleocapsids. Knowledge regarding how large size nucleocapsids pass through the compact nuclear lamina and double-layered nuclear membranes for subsequent cytoplasmic maturation is only emerging recently. In the first part of this study, the cellular endosomal sorting complex required for transport (ESCRT) component Alix was found to be recruited to the nuclear rim and periphery, and colocalized with viral BFRF1 protein at nuclear membrane and vesicle-like structures in EBV replicating NA cells. In the transient transfection system, BFRF1 expression alone induces vesicle formation from nucleus-associated membrane by recruiting ESCRT machinery to the nuclear membrane. Expression of the dominant negative form of Vps4, which is the major determinant for ESCRT machinery, or knockdown Alix not only abolishes the BFRF1-induced vesicle formation, but also leads to the accumulation of nucleocapsid proteins in nucleus. Alix is crucial for BFRF1-mediated vesicle formation. Multiple domains within BFRF1 contribute to Alix recruitment, and both Bro and PRR domains of Alix interact with BFRF1, suggesting that BFRF1 recruits the ESCRT components through Alix for the membrane scission during the nuclear egress process of EBV. In the second part of this study, the depletion of free ubiquitin pool by proteasome inhibitor MG132 or expression of viral BGLF4 kinase blocks the BFRF1-induced vesicle formation, indicating the coordination between ubiquitination and phosphorylation may be crucial for BFRF1 mediated functions. Taken together, this study indicates that BFRF1 cooperates with cellular ESCRT machinery to modulate nuclear membrane structure for the nuclear egress of EBV.