Interference of the viral effector proteins pp71 and IE1 with intrinsic and innate immunity against human cytomegalovirus infections

巨细胞病毒调节蛋白pp71和IE1此前已被证明可以改变被称为核结构域10(ND10)或PML核体的细胞亚核结构。ND10代表多种细胞因子的离散染色体间积累，包括hDaxx、Sp100或相扑，它们需要PML蛋白来形成，并作为对抗人类巨细胞病毒和其他病毒的内在免疫的关键介质。当pp71与hDaxx/ATRX相互作用时，IE1与PML结合，导致蛋白质去SUMO。我们先前工作的亮点是发现巨细胞病毒IE1蛋白的晶体结构与TRIM蛋白的螺旋卷曲结构域有显著的同源性。因此，我们可以证明IE1与PML的螺旋卷曲结构域(=TRIM19)以及其他TRIM蛋白如TRIM5pha和TRIM33结合。此外，我们可以证明PML在I型和II型干扰素诱导的基因表达中发挥着新的协同调节作用，这种作用通过IE1的结合而被拮抗。综上所述，这些结果表明IE1不仅调节TRIM19(=PML)在固有免疫和先天免疫中的功能，而且还影响该蛋白家族的其他成员。由于E3连接酶TRIM家族蛋白参与调节由天然免疫模式识别受体触发的多条信号通路，我们有兴趣详细定义哪些TRIM蛋白受IE1调控。此外，IE1与特定TRIM蛋白的卷曲螺旋结构之间的确切相互作用模式仍不清楚。这将通过共结晶和交联研究来研究。基于对相互作用模式的详细了解，我们计划寻找阻断IE1功能从而起到抗病毒作用的多肽和物质。此外，我们检测到不仅IE1，而且pp71还与特定的TRIM蛋白结合，这也将在本项目中进行研究。总体而言，拟议的项目将有助于我们理解IE1和pp71等多功能病毒蛋白如何能够逃避TRIM因子控制的内在和先天免疫机制。

The cytomegaloviral regulatory proteins pp71 and IE1 have previously been shown to modify a cellular subnuclear structure known as nuclear domain 10 (ND10) or PML nuclear bodies. ND10 represent discrete interchromosomal accumulations of multiple cellular factors including hDaxx, Sp100 or SUMO that require the PML protein for their formation and act as key mediators of intrinsic immunity against human cytomegalovirus and other viruses. While pp71 interacts with hDaxx/ATRX, IE1 was shown to bind to PML resulting in protein deSUMOylation. Highlight of our previous work was the finding, that the crystal structure of the cytomegalovirus IE1 protein shares significant homology to the coiled-coil domain of TRIM proteins. Consequently, we could show that IE1 binds to the coiled-coil domain of PML (=TRIM19) and also other TRIM proteins like TRIM5alpha and TRIM33. Furthermore, we could demonstrate that PML plays a novel co-regulatory role in type-I and type-II interferon-induced gene expression which is antagonized via the binding of IE1. In summary, these result suggest that IE1 not only modulates functions of TRIM19 (= PML) in intrinsic and innate immunity but also affects additional members of this protein family. Since the E3 ligase TRIM family of proteins is implicated to regulate multiple signaling pathways triggered by innate immune-pattern-recognition receptors, we are interested to define in detail which TRIM proteins are modulated by IE1. Furthermore, the exact mode of interaction between IE1 and the coiled-coil structures of specific TRIM proteins is still unclear. This will be investigated by co-crystallization and by crosslinking studies. Based on detailed knowledge of the interaction mode we plan to search for peptides and substances that block IE1 function and thus act antiviral. Furthermore, we detected that not only IE1 but also pp71 binds to specific TRIM proteins, which will also be investigated during this project. Overall, the proposed project will foster our understanding how multifunctional viral proteins like IE1 and pp71 are able to evade intrinsic and innate immune mechanisms governed by TRIM factors.