Impact of herpesvirus protein kinases on host protein modification

Epstein-Barr病毒（EBV）蛋白激酶BGLF4是保守的疱疹病毒蛋白激酶的一员，这是一组在疱疹病毒科所有亚家族中保守的酶。了解BGLF4的细胞蛋白底物对于理解BGLF4的功能至关重要；然而，迄今为止，只有少数宿主底物被表征。除了诱导蛋白磷酸化外，BGLF4还以激酶活性依赖的方式调节宿主蛋白SUMOylation；然而，目前还没有BGLF4调控宿主蛋白的具体例子。为了了解EBV感染给细胞环境带来的变化，不仅需要确定BGLF4在体内的靶标及其精确的磷酸化位点，而且需要一种能够明确解剖BGLF4调控的复杂信号网络的方法。我们之前鉴定了几百个被EBV BGLF4和其他三种同源激酶磷酸化的宿主底物。对它们共享底物的生物信息学分析显示，参与DNA损伤反应（DDR）途径的蛋白质在统计学上是丰富的。进一步的研究表明，BGLF4通过TIP60磷酸化积极诱导宿主DDR，促进病毒复制。有趣的是，我最近的工作表明，小泛素样修饰物（SUMO）的结合对于bglf4诱导的DDR中涉及的蛋白质磷酸化至关重要。BGLF4也以SUMO结合和激酶活性依赖的方式抑制宿主蛋白的SUMO化。基于这些观察，我假设EBV BGLF4诱导细胞蛋白磷酸化和sumo化的动态改变，为病毒的有效复制创造合适的环境。为了准确识别BGLF4下游的信号事件，我将与我的共同导师Akhilesh Pandey博士合作，他是质谱，蛋白质组学和生物信息学领域的领先科学家。我的主要导师戴安·海沃德博士拥有超过30年的EBV研究经验，她将指导我对宿主底物的验证，并展示它们在EBV生物学中的作用。具体来说，在K99期，我将在ebv感染细胞中全球鉴定BGLF4调控的磷酸化和SUMOylation靶点，查明BGLF4靶向的关键宿主途径，并验证鉴定的底物。在R00期，我将阐明BGLF4介导的宿主蛋白磷酸化和SUMOylation的变化促进病毒复制的机制。在这个申请的指导阶段，我提出的研究是病毒学、蛋白质组学和生物信息学等多学科训练的理想载体，所有这些都将确保我在随后的独立阶段取得成功。这些研究的新颖之处在于，它们探索了病毒蛋白BGLF4在调节蛋白磷酸化和sumo酰化之间的串扰中的积极作用。它们还将显著增加我们对病毒操纵宿主磷酸化和SUMOylation的理解，这将导致对病原体-宿主相互作用的新见解。eb病毒（EBV）引起传染性单核细胞增多症，并与几种人类癌症有关。EBV表达一种蛋白激酶BGLF4，它控制着病毒生命周期的多个关键步骤，是抗病毒药物开发的靶标。我提出了一种使用高通量质谱分析BGLF4调节的复杂信号网络的新策略。

DESCRIPTION (provided by applicant): The Epstein-Barr virus (EBV) protein kinase BGLF4 is a member of the conserved herpesvirus protein kinases, a group of enzymes conserved throughout all subfamilies of Herpesviridae. Knowledge of the cellular protein substrates of BGLF4 is essential for understanding BGLF4 function; however, only a small number of host substrates have been characterized to date. In addition to inducing protein phosphorylation, BGLF4 also regulates global host protein SUMOylation in a kinase activity dependent manner; however, no specific examples of a BGLF4 regulated host protein have been described. To understand the changes brought about in the cellular environment by EBV infection, it is not only critical to identify the BGLF4 in vivo targets and their precise phosphorylation sites, but alo to have an approach that can unambiguously dissect the complex signaling networks regulated by BGLF4. We previously identified several hundred host substrates phosphorylated in vitro by EBV BGLF4 and by three other orthologous kinases. Bioinformatic analysis of their shared substrates revealed that proteins involved in the DNA damage response (DDR) pathway were statistically enriched. Further studies demonstrated that BGLF4 actively induces a host DDR via TIP60 phosphorylation to foster viral replication. Interestingly, my recent work demonstrated that binding of the small ubiquitin-like modifier (SUMO) is critical for the BGLF4-induced protein phosphorylation involved in the DDR. BGLF4 also inhibits host protein SUMOylation in both a SUMO binding and kinase activity dependent manner. Based on these observations, I hypothesize that EBV BGLF4 induces a dynamic alteration of cellular protein phosphorylation and SUMOylation to create a suitable environment for efficient virus replication. In order to precisely identify the signaling events downstream of BGLF4, I will work with my co-mentor Dr. Akhilesh Pandey, who is a leading scientist in mass spectrometry, proteomics and bioinformatics. My primary mentor Dr. Diane Hayward, who has over 30 years' experience with EBV, will guide my validation of the host substrates and demonstration of their role in EBV biology. Specifically, during the K99 phase, I will globally identify BGLF4-regulated phosphorylation and SUMOylation targets in EBV-infected cells, pinpoint the key host pathways targeted by BGLF4, and validate the identified substrates. In the R00 phase, I will elucidate the mechanisms by which BGLF4 mediated changes in phosphorylation and SUMOylation of host proteins facilitate virus replication. My proposed studies during the mentored phase of this application are an ideal vehicle for multidisciplinary training in virology, proteomics and bioinformatics, all of which will ensure my future success in the subsequent independent phase. The proposed studies are novel in that they explore the role of a viral protein BGLF4 as an active player in regulating the crosstalk between protein phosphorylation and SUMOylation. They will also significantly increase our understanding of viral manipulation of host phosphorylation and SUMOylation, which should lead to novel insights into pathogen-host interactions. Epstein-Barr virus (EBV) causes infectious mononucleosis and is implicated in several human cancers. EBV expresses a protein kinase, BGLF4, that controls multiple critical steps in the viral life cycle and is a target for anti-viral drug development. I propose a novel strategy using high throughput mass spectrometry to dissect the complex signaling networks regulated by BGLF4.