The Induction of Interferon Regulatory Factor 7 Sumoylation by EBV LMP1

Bentz, Gretchen

Abstract

Epstein-Barr virus (EBV) is a tumor causing virus that is associated with several types of cancer, and the main EBV oncoprotein is latent membrane protein (LMP) 1, a constitutively active membrane protein that mimics the signaling induced by the tumor necrosis factor receptor family. We have reported that LMP1 induces the expression of interferon regulatory factor (IRF)-7, a transcription factor that is a key regulator of Type I interferons in innate immune responses. LMP1 regulates the transcriptional activity of IRF7 via post-translational modifications, including phosphorylation and ubiquitination. Therefore, we investigated if LMP1 induces other modifications of IRF7, specifically that by the small ubiquitin-like protein modifier (SUMO)-1, in order to regulates its function. We found that LMP1 interacts with Ubc9, the only reported SUMO-conjugating enzyme, possibly via the understudied C-terminal activating region (CTAR3). LMP1 also induced the interaction of Ubc9 with IRF7 leading to the sumoylation of IRF7, which resulted in decreased IRF7 turnover. Sumoylated IRF7 accumulated in the nucleus where its transcriptional activity is modestly increased. Therefore, we hypothesize that the induction of the sumoylation of IRF7 by LMP1 moderates the activation of the immune response, and we propose two aims to test this hypothesis. First, we will determine the requirements for IRF7 sumoylation. Several IRF7 deletion and point mutants, where lysines are mutated to arginines, will be examined to determine which lysine residue(s) is/are sumoylated. In addition, a LMP1 CTAR3 deletion mutant will be examined for its ability to sumoylate IRF7. Finally, we will examine the role IRF7 sumoylation has on the induction of innate immune responses. The results will provide insight into how IRF7 is sumoylated and implicate a new function for understudied LMP1 CTAR3. Second, we will investigate competition between sumoylation and ubiquitination of IRF7. Each post- translational modification can alter IRF7 function in a distinct manner, so we will investigate the role sumoylation has on IRF7 ubiquitination, and vice-versa. The effect these modifications have on IRF7 transcriptional activity will also be examined to decipher how the different modifications alter IRF7's function as a central transcription factor for innate immune responses. Relevance to public health: Regulating protein sumoylation is a new target for anti-cancer therapies. This work will further our understanding of protein sumoylation during EBV latency, by defining a new role for LMP1 CTAR3 and deciphering how sumoylation affected the function of IRF7. Together, this project will provide insight into new role for LMP1 and IRF7 during EBV latency as well as potential targets for the regulation of anti-viral immune responses.

Public Health Relevance

Regulating the modification of proteins is a new target for anti-cancer therapies. The work proposed here examines how a viral protein induces the modification of a cellular protein in order to regulate immune responses. This work will provide potential targets for the treatment of cancers associated with the virus and the regulation of anti-viral immune responses.