EB病毒BGLF4蛋白質激酶對細胞週期調控之探討

Abstract

病毒為了能在感染宿主細胞後成功複製基因體並組裝病毒粒子，演化出許多策略。過去關於疱疹病毒科的病毒研究中發現，最重要的機制之一就是調控宿主細胞的細胞週期，造成宿主細胞內一個類似細胞週期中S-phase的環境，但細胞DNA複製受到抑制，以提供足夠的複製原料供病毒使用。BGLF4是EB病毒所帶有的Ser/Thr蛋白質激酶，目前已知具有類似CDK的活性。當BGLF4單獨表現於細胞時，會引起未成熟的細胞染色體濃縮、核板蛋白層溶解、細胞骨架重組等未成熟細胞分裂的現象，也會抑制DNA旋解酶複合體MCM4-MCM6-MCM7中MCM4次單元體之活性，進而抑制宿主細胞DNA複製。然而BGLF4是否可直接調控細胞週期的進行則尚未釐清。本研究中，我們利用293 T-REx誘導細胞株發現，BGLF4會造成細胞生長遲緩，利用表現量不同的細胞株進行實驗還發現此現象與BGLF4表現量有正相關性。此外，BGLF4也會影響細胞週期S-phase的進行，使細胞延緩進入G2/M-phase的時間點。若利用HeLa細胞將GFP-BGLF4轉染送入細胞後，對BGLF4表現量多或表現量少的細胞群各別分析其DNA含量，則發現BGLF4表現量高時S-phase細胞明顯增加，而且這些細胞的細胞週期將無法離開S-phase往G2/M-phase過渡轉換時期推進。最後利用動態曠時攝影，以GFP-H2B-HeLa穩定細胞，在外送DsRed-BGLF4後，針對單一表現BGLF4的細胞進行觀察。發現若BGLF4表現低，未引起或僅引起輕微未成熟染色體濃縮，細胞可以完成細胞週期，分裂成兩顆子細胞。若BGLF4表現時細胞正好處於M-phase、具有濃縮的染色體，有絲分裂也可以順利進行。但如果還在interphase即大量表現BGLF4、並引發嚴重的未成熟染色體濃縮，細胞不但無法完成DNA複製完成細胞週期，進一步還會造成不正常的染色體結構，引發細胞凋亡的訊息。綜合以上實驗結果，我們發現BGLF4會影響細胞週期的進行，但是單一細胞的結果會由於BGLF4表現時所處的細胞週期時間點與BGLF4蛋白質的表現量而有不同的命運。

To optimally use host cell environment for viral replication and virion production, viruses have evolved several approaches. In the instance of herpesviruses, one of the most elegant strategies is to manipulate host cell cycle to create an S-phase like environment, interfere cellular DNA synthesis, and thus provide adequate materials for viral replication. BGLF4 is a proline-dependent Ser/Thr protein kinase encoded by Epstein-Barr virus (EBV) and is considered to possess CDK-like activity. In previous studies, BGLF4 was uncovered to induce unscheduled chromosome condensation, leading to premature mitotic events and block MCM complex activity. However, it is still unknown whether BGLF4 directly regulates cell cycle. In the first part of this study, we used 293 T-REx BGLF4 inducible cells and discovered that expression of BGLF4 results in cell growth retardation, and interferes with S-phase progression, leading to G2/M-entry delay. These effects are clearer in clones with higher BGLF4 expression. In HeLa cells trasnfected with GFP-BGLF4 expressing plasmids, cells were sorted into two groups with various levels of BGLF4 expression by FACS. We found that these two populations of cells display dramatically different cell cycle profile. Higher BGLF4 expression leads to an S-phase accumulation, but lower BGLF4 expression virtually hast no such effect. Furthermore, in the time-lapse vedio observation, we found that cells with only slightly BGLF4-induced premature chromosome condensation can complete cell cycle. If BGLF4 initially expresses at M-phase, cells can also divide. On the other hand, abnormal chromosomal structure and apoptotic signaling were uncovered in high-BGLF4-expressing cells. Together, these findings suggest that BGLF4 interferes with cell growth and S-phase progression, however, individual cell fate depends on the levels and timing of BGLF4 expression.