PROJECT 3 – PROJECT SUMMARY/ABSTRACT Project 3. Latent and Lytic EBV Infection in Epithelial Cells Shannon Kenney, Project Leader; Eric Johannsen, Co-Leader Epstein-Barr virus (EBV) is an important cause of human epithelial cell cancers, particularly undifferentiated nasopharyngeal carcinoma (NPC), but the precise mechanism(s) by which latent EBV infection promotes NPCs are poorly understood. EBV also induces oral hairy leukoplakia (OHL) lesions in the tongue epithelium of AIDS patients, which is caused by completely lytic EBV infection in differentiated squamous epithelial cells. We recently discovered that latent EBV infection inhibits differentiation, and promotes proliferation, of a telomerase- immortalized normal oral keratinocyte cell line (NOKs), and that this effect is mediated by the latent EBV protein, LMP1. Our exciting preliminary results show that LMP1 affects NOKs proliferation and differentiation by activating the downstream targets of the Hippo tumor suppressive pathway, YAP and TAZ. Conversely, we have discovered that the ability of EBV to enter lytic viral infection in NOKs is induced by cellular differentiation and requires the differentiation-associated proteins, IRF6/RIPK4, as well as KLF4/BLIMP1/YAP/TAZ, although the pathways that regulate lytic EBV reactivation during differentiation are not yet fully defined. In Project 4, a collaboration between the Kenney and Johannsen labs, we will dissect how the latent EBV oncoprotein LMP1, in conjunction with YAP/TAZ, inhibits epithelial cell differentiation and enhances proliferation (both key steps in the pathway to tumor formation), and further define how lytic EBV reactivation is activated by differentiation- dependent mechanisms in normal epithelial cells. In Aim 1, we will define how LMP1 activates YAP and TAZ, and inhibits cellular differentiation, in NOKs. In Aim 2, we will identify novel cellular genes/pathways that drive or inhibit lytic EBV in NOKs. In Aim 3, we will ask if FDA-approved drugs that inhibit or activate YAP/TAZ activity block the growth of EBV+ epithelial cell tumors and/or regulate lytic EBV reactivation. We hypothesize that LMP1 inhibits differentiation by activating YAP and/or TAZ (Aim 1), that epithelial cell differentiation induces lytic EBV reactivation at least partially though activation of the RIPK4/IRF6 axis (Aim 2), and that YAP/TAZ inhibitors will impair the growth of EBV-infected NPCs and gastric cancers expressing LMP1 (Aim 3). This project involves collaborations between multiple investigators, including Dr. Kenney, Dr. Johannsen and Dr. Lambert, and will use multiple Cores, including the virus core, the microscopy core, and the administrative core.