The recent article by Nikitin et al
provides new insight into how the host DNA damage response (DDR) restricts Epstein-Barr virus (EBV)-induced B cell transformation and how EBV overcomes this barrier to establish latency [1
]. EBV was first discovered in 1964 in cultured lymphoblasts derived from a Burkitt’s lymphoma [2
]. EBV infects 90%–95% of world’s population and is now known to contribute to a variety of human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, Burkitt’s lymphoma, and post-transplant lymphoproliferative disease [3
]. The life cycle of EBV includes both latent and lytic cycles, which are dependent on distinct programs of viral gene expression. During the EBV latent cycle, a very limited set of viral proteins are produced. The viral genomes are maintained as circular episomes and there is no production of virions. In the latent state, viral DNA is replicated only once during S phase, in synchrony with the host chromosomal DNA[4
]. Lytic cycle infection is characterized by sequential expression of the bulk of the viral genome and massive viral DNA replication with the ultimate production of infectious virions [5
]. It is recognized that viral latency gene products drive oncogenesis. However, recent studies have also implicated lytic EBV infection in the development of lymphomas [7
]. Therefore, both latent and lytic replication may contribute to the emergence of cancers.
Previous studies showed that the DDR plays an important role in the viral life cycle. EBV lytic replication elicits a DDR by triggering ATM autophosphorylation and activation. Activated ATM phosphorylates its downstream targets, such as H2AX, p53, CHK2 and RPA2. In addition, phosphorylated ATM, RPA2 and Mre11/Rad50/Nbs1 (MRN) complexes are recruited to replication compartments in nuclei during EBV lytic replication [9
]. In the early stages of the EBV lytic cycle, ATM mediated phosphorylation of p53 at Ser15 prevents its MDM2-dependent degradation and promotes its function in the DDR. However, during the later stages, CHK2 may contribute to p53 phosphorylation at Ser366 and Ser378, resulting in p53 degradation through the ubiquitin-proteasome pathway and blocking of p53-mediated apoptosis [11
] and the EBV protein kinase BGLF4 contributes to p27 phosphorylation and its ubiquitin-proteasome dependent degradation [12
]. These events lead to a prolonged pseudo S-phase environment that fosters viral DNA replication (). Like EBV, other viruses, such as herpes simplex virus (HSV) [13
], parvovirus minute virus of mice (MVM) [14
] and murine γ-herpesvirus 68 (MHV 68) [15
] also trigger the DDR to benefit viral replication.
Differing roles of the DNA damage response in promoting viral replication and restricting latency in EBV-infected cells
To better understand the host response to viral infection that limits EBV-induced transformation, Nikitin et al.
performed a detailed study using EBV infection of primary B cells [1
]. First, they found that EBV infection of these cells elicits the DDR, and EBNA2 and latency III gene products are necessary to induce the response. Unexpectedly, the EBV-induced DDR is independent of the presence of nuclear viral genomes and of lytic replication while it is associated with a transient hyperproliferation of the host cell. Further, mRNA microarray analysis revealed that genes induced during this transient hyperproliferation period are enriched in “Cell proliferation” and “Response to DNA damage stimulus” categories, and are also consistent with ATM-dependent p53 target gene expression. The activated genes were subsequently repressed in the transition from the transient hyperproliferation stage to the indefinitely proliferating stage (lymphoblastoid cell line [LCL] outgrowth). The transition in cell gene expression correlates with the shift of viral gene expression from early Cp promoter usage and high EBNA2 expression in proliferating cells to Wp promoter usage and the reduced EBNA2 expression in established LCLs. Cells in early hyperproliferating divisions are more prone to growth arrest and cell death than those in later divisions, supporting the hypothesis that the EBV-induced DDR is growth suppressive.
Since ATM and its downstream effector kinase CHK2 are critical kinases involved in the DDR, blocking these kinases by inhibitors was predicted to remove the DDR restriction on B cell proliferation. Indeed, Nikitin et al. found that the efficiency of EBV-mediated transformation of peripheral blood mononuclear cells increased with treatment by small molecule inhibitors of either ATM or CHK2. The inhibitor treatment is most effective during the hyperproliferation period, suggesting that the EBV induced ATM- and CHK2-dependent growth suppressive signaling pathway restricts transformation efficiency. In order to establish latency, EBV must evolve a strategy to overcome the inhibiting effects of the host DDR. In this study, Nikitin et al. further demonstrate that EBNA3C is required to attenuate the DDR and facilitate immortalization.
Previously published data had emphasized EBV utilization of the host DDR to promote viral replication. Nikitin et al.
however demonstrate that EBV-induced DDR is a robust host anti-viral strategy, consistent with the important role of the DDR as a tumor suppressor pathway during oncogenesis [16
]. Therefore, the DDR is differentially regulated during different stages of EBV’s life cycle, illustrating the complexity of the EBV-host interaction. Other viruses have similarly evolved complex relationships with the host DDR [18
]. Kaposi’s sarcoma associated herpesvirus (KSHV) encodes vIRF1 that targets ATM and p53 to counteract the DDR and facilitate viral replication [19
]. The DDR induced by KSHV v-cyclin also functions as an anti-cancer barrier in KSHV-induced cancers [20
]. Human papillomavirus E6 binds to XRCC1 and inhibits its function in DNA repair and genomic stability [21
], and E6 also destabilizes TIP60, the upstream regulator of ATM and the DDR [22
], which allows HPV to promote cell proliferation and cell survival [24
]. MHV68 activates the DDR through H2AX phosphroylation to foster viral lytic replication [15
], while H2AX knockdown also reduces establishment of latency [25