Epstein-Barr virus (EBV) BZLF1 protein interferes with cellular response to DNA damage

Abstract

The Epstein-Barr virus (EBV) is a human gamma herpesvirus. EBV infection is associated with various kinds of cancers including B cell and epithelial cell malignancies notably the nasopharyngeal carcinoma (NPC) and gastric carcinoma. The EBV immediate-early protein, BZLF1, is the key mediator that switches EBV infection from latent to lytic stage. The BZLF1 induces cell cycle arrest, however, the effects appear to be cell-type dependent. The involvement of BZLF1 on cell cycle progression in NPC cells has not been well-defined. DNA damage response (DDR) pathways induce cell cycle checkpoint arrest and serve as a surveillance mechanism to protect mammalian cells from DNA damage. A functional DDR pathway is essential for the maintenance of genomic integrity in cells. Interestingly, EBV lytic cycle has been shown to be intimately linked to DDR pathways. The BZLF1 has been shown previously to be a binding partner of several DDR proteins. In this study, the impact of BZLF1 on cell cycle was examined in NPC cells (HONE1). Expression of BZLF1 induced cell cycle arrest at G1, which is more prominently demonstrated after treatment with nocodazole. The cell cycle arrest is associated with up-regulation of p21 and p27, as well as down-regulation of cyclin B1. The arrest of cells in G1 phase by BZLF1 may provide a more favorable cellular environment to facilitate lytic EBV replication in infected cells. Furthermore, expression of BZLF1 was shown to impair DDR pathways in HONE1 cells, particularly in blocking the recruitment of 53BP1 to IRIF (ionizing radiation induced foci) induced by DNA damage. The expression of BZLF1 impairs the binding between two crucial DNA damage response proteins, RNF8 and MDC1, which in turn impairs localization of both RNF8 and 53BP1 to the ionizing radiation induced foci. Further functional studies showed that BZLF1 impairs DNA damage repair and abrogates G2/M checkpoint in cells rendering them more sensitive to irradiation and induce genomic instability. Moreover, the blockage of 53BP1 and RNF8 foci formation was also observed, though to a lesser extent, in EBV-infected cells induced to undergo lytic infection. These findings indicate that activation of BZLF1 in EBV-infected NPC cells induces DNA damage and preferentially sensitizing NPC cells to irradiation and/or chemo-treatment. These observations reveal a novel mechanism on how lytic EBV infection may induce genomic instability in cells and have direct implication on the role of EBV infection and NPC pathogenesis. Bmi1 is a transcriptional repressor highly expressed in NPC cells. It has been shown to be involved at the early stage of DDR response. Moreover, it also renders resistance to numerous types of stress-induced cytotoxicity in several cancer cell lines. In NPC cells, overexpression of Bmi1 confers resistance to IR-induced apoptosis. However, preliminary results did not reveal a significant involvement of Bmi1 in DDR in NPC cells. Further investigations are warranted to elucidate the involvement of Bmi1 in DNA damage in NPC cells.