Cellular Mechanism of Reactivation of Lytic Cycle of Epstein-Barr Virus (EBV) by a Novel Compound, C7, in EVV-associated Epithelia Malignancies

Abstract

Introduction and Project Objectives: Pharmaceutical reactivation of Epstein–Barr virus (EBV) lytic cycle represents a potential therapeutic strategy against EBV-associated epithelial malignancies such as gastric carcinoma and nasopharyngeal carcinoma (NPC). A novel lytic-inducing compound, C7, which exhibits structural similarity to Dp44mT, a known intracellular iron chelator, was found to reactivate EBV lytic cycle in GC and NPC. This study aims to 1) delineate the mode of action of C7 and clinically available iron chelators in EBV lytic reactivation, and 2) to determine the cytopathic effect and synergism on EBV lytic reactivation by combining C7 with ganciclovir or other lytic inducers that possess distinct mechanism in EBV lytic reactivation such as histone deacetylase inhibitor (HDACi). Methods and Results: Previous study showed the activation of the hypoxia signaling pathway upon C7/iron chelator treatment. We verified that the hypoxia signaling pathway was not the only pathway associated with EBV lytic reactivation induced by C7/iron chelators. Treatment with either the ERK1/2 or autophagy inhibitor significantly abolished C7-mediated EBV lytic reactivation but not in those induced by HDACi, suggesting the involvement of the ERK1/2-autophagy pathway in EBV lytic cycle reactivated only by C7/iron chelators. In addition, these two subclasses of lytic inducers imposed different cellular effects and led to distinct stages of cell cycle arrest in NPC cells. Furthermore, only the inhibition of autophagy initiation was required for EBV lytic reactivation. siRNA knockdown of various autophagic proteins of the early autophagy stages such as beclin-1, ATG3, ATG5, ATG7, LC3B, ATG10, AT12 and Rab9, revealed only the knockdown of ATG5 diminished EBV lytic reactivation, indicating a specific role of ATG5 in C7-reactivated EBV lytic cycle. Conclusion: This study has introduced C7 and clinically available iron chelators as a new class of compounds for EBV lytic reactivation. They reactivate the viral lytic cycle through intracellular iron chelation and the activation of the ERK1/2-autophagy axis, which represent novel and distinct mechanism from that of the conventional lytic inducers. This supports the introduction of C7/iron chelators to the conventional drug reservoir to enhance the efficacy and explore possible drug combination in the lytic induction therapy against EBV-positive malignancies.