人疱疹病毒8型复制与SUMO化修饰之间的相互作用及机制研究

Post-translational modification by small ubiquitin-like modifier (SUMO), termed SUMOylation, has emerged as a rapid and dynamic regulator of protein localization, stability and activity. SUMOylation controls a variety of important cellular processes, including signal transduction, replication, chromosome segregation, DNA repair, and is therefore a preferential target for viral attack. Human herpesvirus 8 (HHV-8) seems to have evolved mechanisms that directly or indirectly modulate the SUMO machinery in order to evade host immune surveillance, thus advancing their survival. Interestingly, HHV-8 encodes RTA (replication and transcriptional activator), a SUMO-targeted ubiquitin E3 ligase (STUbL), and K-bZIP (KSHV-encoded basic leucine zipper protein), a SUMO E3 ligase, which functions oppositely. We hypothesize that at the initial stage of viral replication, RTA activates K-bZIP, up-regulates the global SUMOylation level to repress host immune-related genes. However, subsequently, after the accumulation of viral early products, RTA starts to degrade K-bZIP, down-regulates the level of SUMOylation, so that viral late genes can be expressed and virion assembly can be accomplished. It is likely that as the only switch protein to trigger viral reactivation, RTA controls the replication schedule probably through modulating host SUMOylation. To validate this hypothesis, in this proposal, HHV-8 reactivation system and the de novo infection system will be utilized to investigate 1) the effects of HHV-8 replication on the cellular global SUMOylation, and on the level of SUMO-catalyzing enzymes; 2) the mechanism of RTA activation and degradation of K-bZIP and their roles in the modulation of cellular global SUMO levels; 3) the dependence of different viral promoters on the repression of K-bZIP and on the RTA-modulated SUMOylation levels; and 4) the effects of cellular global SUMOylation on the HHV-8 replication. The objective of this proposal is to establish the model that RTA controls the schedule of viral replication via its dynamic regulation of K-bZIP and the cellular SUMOylation level. This study will contribute to the understanding of the interaction between HHV-8 replication and host SUMOylation, and will facilitate the development of new strategies to interrupt viral replication.

SUMO化修饰调节蛋白的活性和定位，控制生理活动及抗病毒反应，是病毒优先攻击的目标之一。病毒复制可改变SUMO化水平。人疱疹病毒8型（HHV-8）编码SUMO引导的泛素连接酶RTA和SUMO连接酶K-bZIP，双向调节SUMO化修饰。我们推测RTA在复制早期激活K-bZIP以提升SUMO化水平，随后降解K-bZIP导致SUMO化水平骤降而开启晚期基因并组装毒粒，凭RTA两度逆转SUMO化水平实现对病毒裂解复制的时序控制。拟利用HHV-8重激活和感染模型，研究：1）病毒复制对宿主SUMO化水平和修饰酶的影响；2）RTA激活和降解K-bZIP对SUMO化修饰的调节；3）病毒不同启动子对K-bZIP抑制和SUMO化水平依赖性的差异。阐明HHV-8复制对宿主SUMO化修饰的影响，揭示宿主SUMO化水平的波动与病毒基因表达时序间的关系，初步建立RTA通过控制宿主SUMO化水平调节病毒复制的时序模型。

小类泛素化修饰（SUMOylation）能调节被修饰蛋白的亚细胞定位、稳定性和活性，参与很多重要生理活动的调节，也是病毒优先盗取的目标。人疱疹病毒8型（HHV-8），又称卡波西肉瘤相关疱疹病毒（KSHV），是重要的HIV相关病毒，其导致的恶性肿瘤是众多艾滋病患者的死亡原因。HHV-8是第一个被发现同时编码靶向SUMO的泛素连接酶（STUbL）和SUMO连接酶的病毒，其orf50基因编码的复制与转录激活蛋白（RTA）具有STUbL活性；k8基因编码的K-bZIP蛋白具有SUMO连接酶活性。在本项研究中，我们证实了转染RTA真核表达质粒可使293T细胞中被SUMO化修饰的蛋白明显减少；而转染K-bZIP真核表达质粒则可增加被SUMO化修饰的蛋白。利用萤火虫荧光素酶报告系统和免疫印迹实验，我们解析了RTA对k8的双向调节机制，发现RTA既能激活k8启动子，又能以依赖26S蛋白酶体的途径降解K-bZIP蛋白。借助TRE×BCBL-1-RTA 四环素诱导系统，我们研究了重激活过程所伴随的病毒蛋白表达的时序特点和细胞整体SUMO化水平的变化，发现细胞中被SUMO化修饰的蛋白在重激活后1小时开始增多，持续到24 小时之后才显著下降，这与RTA在重激活后24 小时达到峰值，而K-bZIP在8 小时达到峰值后又急剧减少的结果相吻合，说明RTA不仅作为STUbL直接调节细胞整体SUMO化修饰水平，还可通过对SUMO连接酶K-bZIP的双向调节，在病毒重激活过程中，实现对宿主细胞整体SUMO化修饰水平的精细调控。作为控制病毒从潜伏到裂解转换的分子开关，RTA可激活很多下游病毒基因的表达，我们比较了RTA激活不同病毒启动子受SUMO化水平调节的差异。发现SUMO能抑制RTA对orf45和k8的激活，却能增强RTA对早期基因orf57和pan以及晚期基因orf8等的激活。综合上述结果我们提出HHV-8 RTA通过双向调节K-bZIP改变宿主细胞的整体SUMO化水平，并由此实现对下游病毒基因有序开启的模型。本研究为深入理解病毒如何盗用宿主的SUMO化系统调节自身复制进程以及SUMO化修饰在病毒-宿主相互作用中扮演的角色提供了新的证据，为开发新型抗病毒药物和寻找阻断病毒复制的靶点提供了策略和思路。