ASB13 Control of EBV Life Cycle in B Cell and Epithelial Cell Cancers - PROJECT SUMMARY Epstein-Barr virus (EBV), a gamma-herpesvirus, establishes lifelong latency in B cells and contributes to about 2% of human cancers. Presently, specific therapeutic guidelines for managing EBV-associated diseases are lacking, prompting exploration into oncolytic therapies targeting latent virus reactivation for treating EBV-related cancers. Understanding the intricate cellular mechanisms controlling the EBV life cycle is crucial for developing such strategies. In this proposal, we will focus on ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing 13 (ASB13) protein, a member of the ASB family proteins with no known function in viral life cycle. Using CRISPR/Cas9 approaches to screen the role of ASB proteins in EBV life cycle, we found that depletion of several ASB proteins, especially ASB13, significantly promotes EBV replication. We demonstrated that ASB13 interacts with SYK kinase in EBV-positive lymphomas cells to limit EBV replication. Contrary to traditional roles, we found that ASB13 directly inhibits SYK rather than inducing its degradation, sensitizing cells to SYK- dependent caspase activation, facilitating EBV reactivation. Pharmacological inhibition of SYK or caspase suppresses EBV lytic induction in ASB13-depleted cells. Furthermore, we demonstrated that ASB13 also binds and destabilizes the EBV immediate-early protein RTA possibly through its E3 ubiquitin ligase activity, leading to its rapid degradation upon lytic induction. Importantly, we also found that ASB13 is quickly degraded upon lytic induction and SYK inhibition reverses ASB13 degradation, blocking EBV reactivation, indicating a reciprocal regulation between SYK and ASB13. Based on these findings, we hypothesize that ASB13 restricts EBV lytic replication by blocking SYK activation and promoting key viral protein degradation, while its degradation upon lytic induction facilitates viral replication. The specific aims proposed are: Aim 1: To determine the mechanisms by which ASB13 inhibits SYK signaling to restrict EBV replication in B cells. Investigating ASB13's functional regulation of SYK and related signaling molecules in vitro and in BCR activation and EBV lytic replication contexts. Aim 2: To determine the mechanisms by which ASB13 regulates viral protein stability to limit EBV lytic replication. Determining ASB13’s impact on EBV RTA stability, screening for ASB13-interacting proteins among all EBV genes, and assessing its role in viral protein regulation. Aim 3: To elucidate the mechanisms by which ASB13 is degraded to facilitate EBV lytic replication. Investigating ASB13’s mRNA regulation and protein turnover regulated by lytic triggers in EBV-positive cells. Our proposed research will fill the critical knowledge gap regarding ASB13’s role in EBV lytic reactivation, contributing to a deeper understanding of EBV-associated diseases and potential targeted therapies.
