EB病毒BGLF4 蛋白質酶引發未成熟染色體濃縮及核板蛋白層溶解之研究

Study on Epstein-Barr virus BGLF4 kinase-induced premature chromosome condensation and nuclear lamina disassembly

李重霈(Chung-Pei Lee)

指導教授 ： 陳美如

國立臺灣大學/醫學院/微生物學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02055

摘要

泡疹病毒科的病毒在順利感染宿主細胞後，在細胞中面臨複製資源、細胞核內空間及核膜障壁等不同的限制；因此病毒必須演化出一套相應的策略，以進行有效率的病毒基因體複製及病毒顆粒成熟。過去對於EB病毒(Epstein-Barr Virus)複製的研究主要著重於探討參與病毒基因體複製、組裝或細胞週期調控相關病毒及細胞因子；但對EB病毒複製時對於宿主細胞核結構或核中染色質分佈的影響仍無明確及深入的研究。
我的研究首先利用觀察帶有EB病毒的鼻咽癌上皮細胞株NA或B細胞株Akata，發現在病毒再活化(reactivation)的同時，宿主細胞染色質有高度濃縮，而核模呈現不規則外型等現象。進一步探討病毒導致染色質高度濃縮及不規則核模形成的機制，發現EB病毒唯一的Ser/Thr蛋白質激酶BGLF4扮演了重要的角色。當BGLF4激酶單獨表現於細胞時，可在無需細胞DNA複製及細胞分裂蛋白質激酶Cdc2的參與下，造成未成熟染色體濃縮(premature chromosome condensation)、
核板蛋白層溶解(nuclear lamina disassemly)、細胞骨架重組(cytoskeleton rearrangement)等似未成熟細胞分裂現象(premature mitotic events)。在分子機制的探討中發現BGLF4和染色質濃縮主要調控因子濃縮素(condensin)有交互作用。於細胞或試管內實驗發現BGLF4可造成濃縮素於蛋白質激酶Cdc2調控次單元hCAP-G的磷酸化。另一方面，BGLF4可透過和調控細胞DNA結構的第二型拓樸酶
(topoisomerase II)交互作用和磷酸化，進而造成拓樸酶第二型蛋白磷酸化和解DNA環套作用能力(decatenation activity)的增進；顯示BGLF4激酶可能透過活化濃縮素和拓樸酶第二型蛋白造成未成熟染色體濃縮，進而提供更多的染色體外空間(extrachromosomal space)以利病毒複製。此外也發現這個造成宿主細胞未成熟染色體濃縮的能力是存在於其他丙型泡疹病毒激酶(gamma herpesviral kinases)中，如
卡波西氏肉瘤相關性病毒ORF36激酶(KSHV ORF36)及鼠泡疹病毒68型ORF36激酶(murine herpesvirus 68 ORF36)。
由於EB病毒於複製後必須克服來自宿主細胞的核膜障壁，以進行隨後的病毒顆粒成熟。因此在研究EB病毒對於宿主細胞核結構的影響上，發現BGLF4在造成未成熟染色體濃縮的同時也會造成細胞核板蛋白層溶解及細胞骨架重組。BGLF4可以和A和C型板蛋白(lamin A/C)交互作用(interaction)並於試管中造成
A型板蛋白(lamin A)磷酸化；這暗示EBV可透過BGLF4在無須其他病毒蛋白質的協助下直接調節核板蛋白層的完整性，可能進而有助於病毒核心顆粒(nucleocapsid)於細胞核膜的穿透。透過板蛋白點突變試驗，也發現人類泡疹病毒激酶(human herpesviral UL kinases)皆可透過板蛋白上相似的絲胺酸殘基(serine residues)，造成宿主細胞核板蛋白層溶解。綜合本論文的研究發現了一個新的機制，顯示EB病毒BGLF4激酶可透過類似於細胞中Cdc2激酶的作用方式影響不同的細胞受質(substrates)，進而造成多重似未成熟細胞分裂現象，於核內提供更多的染色體外空間及不完整的核板蛋白層。如此可有效解決來自細胞中對於細胞核內空間及核膜障壁的限制，有助於病毒基因體複製及病毒核心顆粒有效率的穿透細胞核膜。推測病毒複製時BGLF4激酶影響造成的宿主細胞核和染色質結構改變，也可能在
EB病毒的致病機轉上扮演角色。

關鍵字

BGLF4 激酶Cdc2 激酶聚合素 ； EB 病毒 ； 板蛋白A/C ； 未成熟染色體濃縮 ； 核板蛋白層溶解 ； 拓樸酶第二型

並列摘要

For efficient virus replication and virion production, herpesviruses must defeat several cellular limitations in replication resources, intranuclear space and nuclear architecture barrier. Previous studies of Epstein-Barr virus (EBV) replication focused mainly on the viral and cellular factors involved in replication compartment and cell cycle control. However, little is known about how EBV reorganizes nuclear architecture, the chromatin territories, nuclear envelope architecture and cell morphology.
In EBV-positive nasopharyngeal carcinoma NA cells or Burkitt’s lymphoma Akata cells, we noticed that cellular chromatin becomes highly condensed and nuclear envelope structure becomes irregular upon EBV reactivation. In searching for the possible mechanisms involved, we found that transient expression of EBV BGLF4 kinase induces unscheduled chromosome condensation and multiple premature mitotic events, such as nuclear lamina disassembly and stress fiber rearrangements, independent of cellular DNA replication and Cdc2 activity. To the mechanism, BGLF4 interacts with condensin complexes and phosphorylates condensin hCAP-G subunit at region similar to Cdc2-targeted. Additionally, BGLF4 phosphorylates and stimulates the decatenation activity of topoisomerase II, suggesting that BGLF4 may induce chromosome condensation through condensin and topoisomerase II activation.
This unscheduled chromosome condensation may provide more extrachromosomal space for virus replication. The ability to induce chromosome condensation is conserved in gamma herpesviral kinase, such as KSHV ORF36 and murine herpesvirus 68 ORF36.
For virion maturation, EBV must overcome the structural barrier of nuclear envelope of host cells. In the study of how EBV regulates nuclear architecture, we found BGLF4 induces the disruption of nuclear lamina and cytoskeleton rearrangement. BGLF4 interacts with lamin A/C and phosphorylates lamin A protein at sites more than Cdc2 targeted, suggesting BGLF4 is able to directly regulate the nuclear lamina independent of other viral proteins. BGLF4 mediated nuclear lamina disassembly may facilitate viral nucleocapsid egress through nuclear envelope. In addition, amino acid Ser-22, Ser-390 and Ser-392 residues of lamin A are important for BGLF4-induced nuclear lamina disassembly. The ability to induce nuclear lamin disassembly is conserved through human herpesviral UL protein kinases. Together, these findings suggest a novel mechanism by which human gamma-herpesviral kinases may induce multiple premature mitotic events to provide more extrachromosomal space and nuclear lamina disassembly for viral DNA replication and successful egress of nucleocapsids. Whether BGLF4 induced architecture changes in cellular chromatin and nuclear lamina may be involved in EBV pathogenesis will need further investigation.

並列關鍵字

BGLF4 kinase ； Cdc2 ； condensin ； Epstein-Barr virus ； lamin A/C ； premature chromosome condensation ； nuclear lamina disassembly ； topoisomerase IIα

參考文獻

Adachi, Y., M. Luke, and U. K. Laemmli. 1991. Chromosome assembly in vitro: topoisomerase II is required for condensation. Cell 64:137-48.

Advani, S. J., R. R. Weichselbaum, and B. Roizman. 2003. Herpes simplex virus 1 activates cdc2 to recruit topoisomerase II alpha for post-DNA synthesis expression of late genes. Proc Natl Acad Sci U S A 100:4825-30.

Aebi, U., J. Cohn, L. Buhle, and L. Gerace. 1986. The nuclear lamina is a meshwork of intermediate-type filaments. Nature 323:560-4.

Aleem, E., H. Kiyokawa, and P. Kaldis. 2005. Cdc2-cyclin E complexes regulate the G1/S phase transition. Nat Cell Biol 7:831-6.

Arita, Y., P. Buffolino, and D. L. Coppock. 1998. Regulation of the cell cycle at the G2/M boundary in metastatic melanoma cells by 12-O-tetradecanoyl phorbol-13-acetate (TPA) by blocking p34cdc2 kinase activity. Exp Cell Res 242:381-90.