RNF4 及 USP11 對 EBV Zta 泛素化修飾的調控

Abstract

EB 病毒 (Epstein-Barr Virus, EBV)，又稱作第四型人類皰疹病毒 (Human Herpesvirus 4, HHV-4)，是在人類首度發現的 DNA 致癌病毒，會感染全球九成以上人口。EBV 的生活史主要分成潛伏期 (Latency) 與溶裂期 (Lytic stage)，在溶裂期時 EBV 會表現由 BZLF1 基因轉錄轉譯出的轉錄因子 Zta 來活化溶裂期基因的表現。Zta 偏好活化啟動子被甲基化修飾的基因表現使 EBV 進入生產循環 (productive cycle)，且 Zta 可以直接結合到複製起始點 oriLyt 並與其它蛋白質形成複合體以活化溶裂期複製。先前研究已指出 Zta 會被接上類泛素 (Small Ubiquitin-like Modifier, SUMO)，被類泛素化修飾的 Zta 其轉錄活性會受到抑制。本研究發現 Zta 的泛素化修飾且其泛素化修飾和泛素-蛋白酶體系統 (ubiquitin-preoteasome system, UPS) 有關。Zta 與 RNF4 會於體內及體外結合。在過量表達 RNF4 的情況下，Zta 的泛素化程度會顯著提高，顯示 RNF4 是 Zta 的泛素連接酶。若將 Zta 上的離氨酸 (lysine) 12位點突變後，Zta 的泛素化程度會因此減少。同時本研究發現在敲落 (knockdown) USP11 基因表現的情況下，Zta的泛素化程度會升高，推測 USP11 與 RNF4 在 Zta 的泛素化修飾上存在拮抗的關係。另外本研究發現過量表現 RNF4 會降低 Zta 在細胞中的穩定性，而 ZK12R 的穩定性並不受影響。最後，敲落USP11 基因表現後會使溶裂期蛋白質的表現量衰減。這些結果顯示 RNF4 與 USP11 在 Zta 泛素化修飾的重要性，且對 EBV 的溶裂期進展有著關鍵性的影響。

Epstein-Barr virus (EBV), also known as Human Herpesvirus 4 (HHV-4), is a human oncovirus which infects 90 percent of global population. The life cycle of EBV can be divided into two stages: latency and lytic cycle. During the lytic stage, EBV expresses Zta, which is a transcription factor encoded by BZLF1, to activate the transcription of lytic genes. Zta preferentially activates the methylated genes, allowing EBV to enter the productive cycle. Zta also binds to oriLyt directly as well as forms the replication complex with other proteins to activate lytic replication. Previous study indicated that Zta is conjugated to SUMOs to repress its transcriptional activity. This study finds that Zta is ubiquitinated and its ubiquitination is involved in ubiquitin-proteasome degradation pathway. Zta also interacts with RNF4 in vivo and in vitro. Overexpression of RNF4 enhanced the ubiquitination of Zta, revealing that RNF4 is an ubiquitin E3 ligase of Zta. Moreover, the levels of ubiquitinated Zta was reduced when lysine 12 residue on Zta is mutated (ZK12R). Additionally, knockdown of USP11 expression increased the levels of ubiquitinated Zta, suggesting that USP11 counteracts the effect of RNF4 on Zta’s ubiquitination. This study also finds that overexpression of RNF4 decreased the stability of Zta, but not that of ZK12R. Finally, knockdown of USP11 gene expression attenuated the expression of lytic proteins. Results of this study suggest the importance of RNF4 and USP11 in the ubiquitination of Zta, which is crucial for the lytic progression of EBV.