USP11對EB病毒Rta及Zta的泛素化所扮演的角色

Abstract

EB病毒（Epstein-Barr Virus）是一種廣泛感染人類的雙股DNA致癌病毒，其生活史分為潛伏期（lysogenic cycle）和溶裂期（lytic cycle），病毒在進入溶裂期時需要Rta和Zta這兩種極早期蛋白質。Rta和Zta 主要作為病毒的轉錄活化因子（transactivator），會透過細胞中的RanBPM進行協同活化（synergistic activation）以幫助活化早期基因使病毒能進行大量的複製。本實驗室先前證實了Rta會被細胞內的一種SUMO-targeted ubiquitin E3 ligase，RNF4，進行泛素化修飾（ubiquitin modification）之後再被降解，因而抑制溶裂期的進展。而USP11是一種會與RNF4拮抗的去泛素化酶（deubiquitinase），本研究室未發表的結果發現USP11可能參與Zta降低Rta的泛素化修飾。本研究透過不會被泛素化的Zta突變蛋白質ZK12R，證明了Zta是利用USP11降低Rta的泛素化修飾而非透過對泛素的競爭。之後以活性突變的USP11蛋白質驗證了USP11 是Rta的去泛素化酶。然後透過抑制基因表現（knockdown）的方式分別確認RanBPM和USP11皆會影響Rta的泛素化修飾，而Zta泛素化修飾則是受USP11影響較大。另外，利用同時過量表現USP11和抑制RanBPM基因表現的方式，驗證了USP11降低Rta泛素化修飾需要有RanBPM參與其中，但是在同時表現Zta和抑制RanBPM表現的情況中，則觀察到RanBPM在Zta降低Rta泛素化修飾的機制中並不是必要的。最後利用穩定抑制USP11表現的P3HR1細胞株，證明缺少USP11會些微抑制溶裂期的進行。綜合以上，本研究發現Zta可以透過細胞內的USP11降低Rta的泛素化修飾，進而穩定Rta，因此有利EB病毒的複製增殖。

Epstein-Barr virus is a double stranded DNA oncovirus. The life cycle of EBV can be separated into latent and lytic cycles. The immediate-early proteins, Rta and Zta are necessary for EBV lytic cycle development. They both are viral transactivators that can synergistically activate viral early genes through RanBPM to facilitate viral DNA replication. Our previous study demonstrates that Rta was ubiquitinated by RNF4, a SUMO-targeted ubiquitin E3 ligase, thereby inhibiting lytic progression. Moreover, USP11, a deubiquitinase, has been shown to counteract the functions of RNF4. Our results also suggest that USP11 participates in the reduction of Rta’s ubiquitination by Zta. Firstly, this study showed that Zta utilized USP11 to reduce the amounts of ubiquitinated Rta, but not through the competition of ubiquitins by using ZK12R, a Zta sumoylation-defective mutant. This study further demonstrated by using a catalytic mutant of USP11 that USP11 is a deubiquitinase of Rta. Moreover, the experiments of gene knockdown confirmed that the ubiquitination of Rta is affected by USP11 and RanBPM. However, Zta’s ubiquitination depends on USP11. Meanwhile, the deubiquitination of Rta by USP11 requires the participation of RanBPM since downregulation of RanBPM inhibites USP11-mediated deubiquitination of Rta. Although RanBPM is necessary in the USP11-mediated Rta deubiquitination, this study found that Zta is able to decrease Rta’s ubiquitination when RanBPM is knocked down by shRNA. This study also showed that EBV lytic cycle was repressed in P3HR1 cells with USP11 silencing. Altogether, this study reveals that Zta is involved in the USP11-mediated deubiquitination of Rta to stabilize Rta to promote EBV lytic development.