Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

To test this idea, Li et al. utilized the human transcription factor (TF) proteome array containing 4191 human proteins to identify commonly shared substrates of herpesvirus-encoded kinases [35] . Parallel kinases assays were performed using the four viral kinases, UL31, UL97, BGLF4 and ORF36, which is encoded by herpes simplex type 1 (HSV1), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) and Kaposi Sarcoma associated-virus (KSHV), respectively [38] . In total, 643 nonredundant substrates were identified across the four kinases and 110 substrates were targets of at least three kinases. GO analysis of the 110 shared substrates indicates that DNA damage functional class was significantly enriched. Among the DNA damage proteins, TIP60 was selected as a lead candidate for regulation of viral replication, due to its roles in DNA damage as well as transcriptional regulation through its HAT activity. Phosphorylation of TIP60 by BGLF4 in EBV-infected B cells was validated during further analysis. BGLF4 is known to phosphorylate multiple EBV proteins and only a small number of host proteins [38, 41] . The functions of its previously-characterized targets are varied, implying that the kinase plays multiple roles to promote viral replication [41] . It is expressed in the early phase of the lytic infection cycle and is localized mainly in the nuclei of EBV-infected cells [42] . BGLF4 knockdown revealed that it is critical for release of infectious virus during viral lytic reactivation [41] . Subsequent experiments demonstrated that BGLF4mediated phosphorylation enhanced TIP60 HAT activity by 10-fold, linking the phosphorylation event to viral replication. They also demonstrated the importance of phosphorylation of host DNA damage proteins for viral replication. More specifically, phosphorylation and activation of TIP60 by BGLF4 triggers EBV-induced DNA damage response (DDR) and promotes positive transcriptional regulation of critical lytic genes involved in viral replication. Lastly, the study confirmed that TIP60 was also required for efficient lytic replication in HCMV, KSHV and HSV-1. Taken together, this unbiased approach provides a novel paradigm for discovery of conserved targets of viral enzymes. While herpes kinases have been credible therapeutic candidates, knowing their targets and the signaling pathways they exploit will better enable the development of widely effective antiviral drugs.