THE ROLE OF SUMOYLATION IN THE EBV LIFE CYCLE
ABSTRACT THE ROLE OF SUMOYLATION IN THE EBV LIFE CYCLE By: ABIGAIL E. HARROD Under the direction of: DR. GRETCHEN L. BENTZ, PHD Epstein-Barr virus (EBV) is responsible for ~1.6% of human cancer cases, world-wide. A hallmark of many EBV-associated cancers and malignancies is an overall increase in cellular protein sumoylation. EBV Latent Membrane Protein 1 (LMP1) is the main viral oncoprotein responsible for dysregulating sumoylation. By directly interacting with enzymes of the sumoylation pathway through its C-Terminal Activating Region 3 (CTAR3) domain, LMP1 dysregulates sumoylation of enzymes and proteins involved in various cell maintenance and signaling pathways, resulting in uncontrolled cell growth and division. LMP1 aids in the maintenance of viral latency through increased sumoylation of the EBV lytic co-repressor, Krab-associated protein-1 (Kap1) and dysregulated sumoylation of innate immune activator, interferon regulatory factor 7 (IRF7). Because EBV, along with other herpes viruses, exploits the SUMO machinery, it has been targeted for viral cancer therapies. Although several natural E1 and E2 inhibitors, including Spectomycin B, ginkgolic acid (GA), anacardic acid, and glycyrrhizic acid (GLA), have been identified, most have low potencies and exhibit toxicity, except for GLA which is relatively non-toxic. Here, we examine the effects of ML-792, a novel synthetic small-molecule inhibitor with a specific binding mechanism, on multiple B-cell lines. We hypothesized that ML-792 would modulate the oncogenic potential of EBV LMP1 by inhibiting sumoylation processes. Western blot analysis revealed that ML-792 decreased global cellular protein sumoylation levels at nanomolar concentrations, while having no effect on ubiquitination. Using Trypan Blue Exclusion Assay, we observed that ML-792 treatment inhibited cell growth, induced cell death, and altered cell-cycle progression. ML-792 decreased the ability of lytic virus to infect new cells and led to increased cell clumping and decreased cell migration following Scratch assays. Measuring EBV DNA levels with qPCR showed that drug treatment induced low levels of viral reactivation in cells. In conclusion, we propose that ML-792 could be a safe and potent treatment option for EBV-induced malignancies.
Harrod, Abigail Elana