2013-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/655002摘要：真核細胞的細胞核膜是一個具有保護基因體功能的高度化結構體，主要由內、外核雙脂膜(INM and ONM) 及內核膜下方的板蛋白複合體 (nuclear lamina) 內襯所構成；在此細胞核膜上，核孔蛋白複合體 (NPCs) 構成連接細胞質與細胞核的孔洞，作為物質運輸及交換使用。在細胞分裂的時期，協調的細胞核膜結構溶解及重組對於子代細胞基因體的完整性更扮演重要的角色。對於大部分需要於宿主細胞核中進行複製的DNA 病毒而言，細胞核膜可以說是一個天然屏障不利於病毒複製。在疱疹病毒DNA 在宿主細胞核中複製完成後，組裝完成的大型病毒核殼體 (115-130 nm)需要進一步的穿越細胞核膜到達細胞質中，以進行進一步的病毒顆粒成熟及修飾。這個過程涉及病毒核殼體對於核膜結構的利用, 稱為初次包膜 (primary envelopement)。在有EBV 複製的細胞中，我們發現細胞核膜產生高度改變，其中包括核孔蛋白及核板蛋白層的重組。此外細胞ESCRT(cellular endosomal sorting complex required for transport) 的組成蛋白也由細胞質中被聚集到細胞核膜上。已知細胞ESCRT 機制參與細胞內膜重組調節的角色；ESCRT-1 及ESCRT-II 蛋白是扮演複合物聚集的功能，而ESCRT-III 可藉由切斷細胞膜細頸部而參與在細胞當中多囊體 (multivesicular body) 形成、細胞分裂及病毒於細胞膜的出芽過程。而對於ESCRT 與細胞核膜的相關性則是一個新發現。在我們先前的研究中發現EB 病毒BGLF4 蛋白激酶可以透過類似細胞CDK (Cyclin-Dependent Kinase) 的活性造成許多類似於細胞分裂前期 (prophase) 的現象，這些變化有助於病毒核殼體穿越核膜。此外兩個EBV 基因產物中可與內核膜作用的BFRF1 及細胞核內存在的BFLF2也扮演重要的角色。雖然BFRF1 及BFLF2 所形成的蛋白複合體可以協助病毒核殼體由細胞核進入細胞質，但詳細機制目前仍不清楚。由於BFRF1 及BFLF2 的蛋白表現量在BGLF4 knockdown的EB 病毒複製細胞中會下降，同時病毒核殼體發現會累積於細胞核內，暗示著BGLF4 也可能參與在BFRF1-BFLF2 複合體調節病毒核殼體穿越核膜的過程中。在本研究計畫中，我們將探討病毒及細胞因子對於 EB 病毒穿越核膜及病毒造成細胞膜結構改變的分子機制。主要的目標為 (1) 研究BGLF4 對於EB 病毒複製時的細胞核模結構變化之作用機制；(2) 探討細胞及病毒因子在進行EB 病毒核殼體初次包膜 (primary envelopment) 時的交互作用；(3) 研究泛素化(ubiquitination)對於BFRF1 在改變核膜結構功能上作用的調節；(4) 分析病毒複製細胞中，病毒於細胞核凹陷處的組裝區域(viral assembly compartment)之蛋白組成。<br> Abstract: The nuclear envelope of eukaryotic cells is a highly organized structure that protects the host genome from cytosolic materials. Nuclear envelope is composed of two layers of lipid-bilayered membranes, termed outer and inner nuclear membrane (ONM and INM) and with a protein lining (nuclear lamina). The nuclear pore complexes (NPCs) form holes on the nuclear envelope, functioning as molecular sieves for molecular transport between cytoplasm and nucleus. The nuclear envelope structure needs to be coordinately disassembled and reassembled at the prophase and telophase of cell cycle to ensure the integrity of genome in daughter cells. For most DNA viruses that need to replicate their genomes in the host nucleus, nuclear envelope is a barrier for the initial infection and also the maturation process of progeny virions. After herpes viral DNA replication and packaged in the nucleus, the largesized nucleocapsids (115-130 nm) needs to be translocated through the nuclear envelope into the cytoplasm for subsequent tagumentation and modification. The nuclear egress of nucleocapsids involves a envelopment process using the nuclear envelope. In cells replicating EBV, we observed that nuclear envelope structure is highly modified; including the redistribution of nucleoporins and lamin A/C. Components of cellular endosomal sorting complex required for transport (ESCRT) are recruited from cytosol to perinuclear space or nuclear rim. The ESCRT machinery is known to catalyze membrane remodeling events. The ESCRT-I and ESCRT-II are responsible for the complex recruitment, whereas the ESCRT-III mediated membrane neck cleavage is important for the biogenesis of multivesicular bodies, cytokinesis and virus budding from cytoplasmic membrane. It is a novel observation that ESCRT in associated with nucleus associated membranes. In our previous studies, we demonstrated that EBV BGLF4 kinase functions through a CDK (Cyclin-Dependent Kinase) mimicry mechanism to induce multiple prophase-like phenomena to facilitate the nuclear egress of nuclear capsids. Two additional EBV gene products also take parts in the nuclear egress process of EBV, namely the inner nuclear membrane associated protein BFRF1 and the intranuclear protein BFLF2. BFRF1 and BFLF2 are able to form protein complexes to translocate nucleocapsids from nucleus into cytoplasm, while detailed mechanism remains to be resolved. In the knockdown of BGLF4, most of nucleocapsids are trapped in the nucleus and the expression levels of BFRF1 and BFLF2 are down regulated in EBV replicating cells, suggesting that BGLF4 also regulates the BRFF1-BFLF2 mediated primary envelopment of EBV. In this study, we intend to study the viral and cellular factors involved in the nuclear egress process of EBV and the molecular mechanism of the virus-induced nuclear envelope structural changes. Specific aims are (1) To study BGLF4 kinase function in inducing nuclear envelope structural changes during EBV replication; (2) To explore the interactions among cellular and viral factors in promoting the primary envelopment of EBV nucleocapsids; (3) To reveal the ubiquitination and BFRF1 function in modifying nuclear envelope structure; (4) To identify the protein components in the viral assembly compartment at the perinuclear concave region.EB 病毒蛋白質激酶鼻咽癌泛素化Epstein–Barr virusprotein kinasenasopharyngyl carcinomaubiquitination