Reuter Nina, Reichel Anna, Stilp Anne-Charlotte, Scherer Myriam, Stamminger Thomas
Institute of Clinical and Molecular Virology, Friedrich Alexander Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany.
Institute of Virology, Ulm University Medical Center, Ulm, Germany.
J Gen Virol. 2018 Mar;99(3):369-378. doi: 10.1099/jgv.0.001021.
The human cytomegalovirus (HCMV) IE2p86 protein is pivotal for coordinated regulation of viral gene expression. Besides functioning as a promiscuous transactivator, IE2p86 is also known to negatively regulate its own transcription. This occurs via direct binding of IE2p86 to a 14-bp palindromic DNA element located between the TATA box and the transcription start site of the major immediate-early promoter (MIEP), which is referred to as the repression signal (CRS). However, the exact mechanism of IE2p86-based autorepression is still unclear. By testing a series of IE2p86 mutants in transient expression assays, we found that not only did a DNA binding-deficient mutant of IE2p86 fail to repress the MIEP, but SUMOylation-negative mutants also failed to repress it. This finding was further supported by infection studies with primary fibroblasts harbouring a MIEP-driven transgene as a reporter. Here, we observed that a recombinant HCMV expressing SUMOylation-negative IE2p86 was defective in transgene downregulation, in contrast to wild-type HCMV. Interestingly, however, a double-mutant virus in which both the SUMO acceptor sites and the SUMO interaction motif (SIM) of IE2p86 were inactivated regained the ability to silence the MIEP. This correlated with increased expression levels of the IE2 isoforms IE2p40 and IE2p60, suggesting that these late proteins may contribute to MIEP suppression, thus compensating for the loss of IE2p86 SUMOylation. In summary, our results show that autorepression of the MIEP is not only regulated by late isoforms of IE2, but also depends on posttranslational SUMO modification, revealing a novel mechanism to fine-tune the expression of this important viral gene region.
人类巨细胞病毒（HCMV）IE2p86蛋白对于病毒基因表达的协调调控至关重要。除了作为一种通用的反式激活因子发挥作用外，IE2p86还已知对其自身转录起负调控作用。这是通过IE2p86直接结合位于主要立即早期启动子（MIEP）的TATA盒和转录起始位点之间的一个14碱基对的回文DNA元件来实现的，该元件被称为抑制信号（CRS）。然而，基于IE2p86的自动抑制的确切机制仍不清楚。通过在瞬时表达实验中测试一系列IE2p86突变体，我们发现不仅IE2p86的DNA结合缺陷突变体无法抑制MIEP，SUMO化阴性突变体也无法抑制它。用携带MIEP驱动的转基因作为报告基因的原代成纤维细胞进行的感染研究进一步支持了这一发现。在这里，我们观察到与野生型HCMV相比，表达SUMO化阴性IE2p86的重组HCMV在转基因下调方面存在缺陷。然而，有趣的是，一种双突变病毒，其中IE2p86的SUMO接受位点和SUMO相互作用基序（SIM）均被灭活，恢复了沉默MIEP的能力。这与IE2亚型IE2p40和IE2p60表达水平的增加相关，表明这些晚期蛋白可能有助于MIEP的抑制，从而补偿IE2p86 SUMO化的缺失。总之，我们的结果表明，MIEP的自动抑制不仅受IE2晚期亚型的调控，还依赖于翻译后SUMO修饰，揭示了一种微调这个重要病毒基因区域表达的新机制。
