Importance of Covalent and Noncovalent SUMO Interactions with the Major Human Cytomegalovirus Transactivator IE2p86 for Viral Infection
Importance of Covalent and Noncovalent SUMO Interactions with the Major Human Cytomegalovirus Transactivator IE2p86 for Viral Infection
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DOI：
10.1128/jvi.01525-09
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发表时间：
2009-12-15
影响因子：
5.4
通讯作者：
Stamminger, Thomas
中科院分区：
文献类型：
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作者：
Berndt, Anja;Hofmann-Winkler, Heike;Stamminger, Thomas
人巨细胞病毒(HCMV)的主要反式激活蛋白IE2p86以前被证明是通过SUMO蛋白的共价连接进行翻译后修饰的，这种修饰发生在位于氨基酸175和180位的两个赖氨酸残基上。受体赖氨酸的突变导致哺乳动物细胞中IE2p86 SUMO化的取消和IE2p86介导的反式激活的强烈减少。在本文中，我们通过酵母双杂交分析，在IE2p86中发现了一个额外的相扑相互作用基序(SIM)，它介导了非共价与相扑的结合。瞬时表达实验表明，IE2p86 SIM突变体的SUMO化显著降低，强烈表明非共价相扑相互作用影响共价相扑偶联的效果。为了确定IE2p86相扑相互作用与病毒复制的相关性，我们制备了来自两个不同的HCMV株(AD169和VR1814)的重组病毒。对表达SUMO化阴性IE2p86病毒的分析显示，由于病毒DNA和蛋白质积累减少，以及即刻早期基因表达的启动减少，病毒复制受到强烈损害。将SIM突变额外引入病毒基因组不会进一步损害病毒复制，但会导致病毒蛋白在感染后的后期表达发生变化。综上所述，这篇论文清楚地表明，IE2p86 SUMO化对于HCMV实验室毒株AD169和临床分离株VR1814的有效复制是必要的，从而对该病毒转录因子的体内功能是必要的。
The major transactivator protein IE2p86 of human cytomegalovirus (HCMV) has previously been shown to undergo posttranslational modification by the covalent attachment of SUMO proteins, termed SUMOylation, which occurs at two lysine residues located at amino acid positions 175 and 180. Mutation of the acceptor lysines resulted in the abrogation of IE2p86 SUMOylation in mammalian cells and a strong reduction of IE2p86-mediated transactivation. In this paper, we identify an additional SUMO interaction motif (SIM) within IE2p86, which mediates noncovalent binding to SUMO, as shown by yeast two-hybrid analyses. Transient-expression experiments revealed that an IE2p86 SIM mutant exhibited significantly reduced SUMOylation, strongly suggesting that noncovalent SUMO interactions affect the efficacy of covalent SUMO coupling. In order to define the relevance of IE2p86 SUMO interactions for viral replication, recombinant viruses originating from two different HCMV strains (AD169 and VR1814) were generated. Analysis of viruses expressing SUMOylation-negative IE2p86 revealed strongly impaired replication due to reduced viral DNA and protein accumulation, as well as diminished initiation of immediate-early gene expression. The additional introduction of the SIM mutation into the viral genome did not further compromise viral replication but resulted in altered expression of viral proteins at late times postinfection. In summary, this paper clearly shows that IE2p86 SUMOylation is necessary for efficient replication of the HCMV laboratory strain AD169 and the clinical isolate VR1814 and thus for the in vivo function of this viral transcription factor.