Role of ATM in the Formation of the Replication Compartment during Lytic Replication of Epstein-Barr Virus in Nasopharyngeal Epithelial Cells

Abstract

Epstein-Barr virus (EBV), a type of oncogenic herpesvirus, is associated with human malignancies. Previous studies have shown that lytic reactivation of EBV in latently infected cells induces an ATM-dependent DNA damage response (DDR). The involvement of ATM activation has been implicated in inducing viral lytic gene transcription to promote lytic reactivation. Its contribution to the formation of a replication compartment during lytic reactivation of EBV remains poorly defined. In this study, the role of ATM in viral DNA replication was investigated in EBV-infected nasopharyngeal epithelial cells. We observed that induction of lytic infection of EBV triggers ATM activation and localization of DDR proteins at the viral replication compartments. Suppression of ATM activity using a small interfering RNA (siRNA) approach or a specific chemical inhibitor profoundly suppressed replication of EBV DNA and production of infectious virions in EBV-infected cells induced to undergo lytic reactivation. We further showed that phosphorylation of Sp1 at the serine-101 residue is essential in promoting the accretion of EBV replication proteins at the replication compartment, which is crucial for replication of viral DNA. Knockdown of Sp1 expression by siRNA effectively suppressed the replication of viral DNA and localization of EBV replication proteins to the replication compartments. Our study supports an important role of ATM activation in lytic reactivation of EBV in epithelial cells, and phosphorylation of Sp1 is an essential process downstream of ATM activation involved in the formation of viral replication compartments. Our study revealed an essential role of the ATM-dependent DDR pathway in lytic reactivation of EBV, suggesting a potential antiviral replication strategy using specific DDR inhibitors. IMPORTANCE Epstein-Barr virus (EBV) is closely associated with human malignancies, including undifferentiated nasopharyngeal carcinoma (NPC), which has a high prevalence in southern China. EBV can establish either latent or lytic infection depending on the cellular context of infected host cells. Recent studies have highlighted the importance of the DNA damage response (DDR), a surveillance mechanism that evolves to maintain genome integrity, in regulating lytic EBV replication. However, the underlying molecular events are largely undefined. ATM is consistently activated in EBV-infected epithelial cells when they are induced to undergo lytic reactivation. Suppression of ATM inhibits replication of viral DNA. Furthermore, we observed that phosphorylation of Sp1 at the serine-101 residue, a downstream event of ATM activation, plays an essential role in the formation of viral replication compartments for replication of virus DNA. Our study provides new insights into the mechanism through which EBV utilizes the host cell machinery to promote replication of viral DNA upon lytic reactivation.