Impact of herpesvirus protein kinases on host protein modification

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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.