Epstein-Barr virus (EBV) is a human herpesvirus which has infected a large majority of the world’s human population. This virus infects epithelial cells of the nasopharynx, where it replicates in a lytic or productive manner, as well as B lymphocytes, where the virus enters a latent state . EBV is associated with a plethora of diseases, including infectious mononucleosis, lymphomas (Burkitt’s lymphoma, Hodgkins lymphoma, post-transplant lymphoproliferative disorder), epithelial-based cancers (including nasopharyngeal carcinoma), multiple sclerosis, and the rare but deadly X-linked lymphoproliferative syndrome . The prevalence of this virus, along with its potential for serious disease, necessitate the study of means to inhibit lytic replication, as well as treatments to kill EBV-positive cancer cells.
EBV lytic replication commences upon infection of new cells, or upon reactivation of the latent virus within cells. During lytic replication the first wave of EBV protein activity includes the immediate-early BZLF1 (Z) and BRLF1 (R) proteins. The Z and R proteins are both absolutely necessary for lytic replication to proceed. These are transcription factors that activate transcription from the promoters of the next wave of EBV genes, the early genes. The early genes encode proteins that act to replicate the viral genome. Lastly, the late genes are expressed to provide the virion structural elements .
The Z and R proteins act not only as transcriptional activators, but also physically interact with several cellular proteins including CREB-binding protein (CBP), in order to promote viral replication in lieu of cellular activities [3, 4]. Z and R have also been found to activate MAPK pathways, including the p38, JNK, and ERK pathways [5–7]. Activation of these pathways has been found to be essential for EBV lytic replication. In addition, both Z and R are SUMO-1 modified, which negatively affects Z transcriptional activity, while enhancing R transcriptional activity [8–11]. A potential means of inhibiting viral replication in cells would be to suppress the activity of Z and/or R. Hindering key viral protein/cellular protein interactions may lead to such suppression of Z and R activities, and therefore inhibit EBV lytic replication.
mTOR is a kinase at the heart of a major signaling pathway. mTOR can be found in two different protein complexes, mTORC1 and mTORC2 [12, 13]. Extracellular signals such as various nutrient levels and growth factors impinge on the mTORC1 pathway to control a variety of processes including protein translation, autophagy, cell growth, and mitochondrial metabolism [12–14]. Pathways that activate mTORC1 include the MAPK ERK and Akt signaling pathways, both of which can be activated by phosphatidylinositol 3 kinase (PI3 kinase) . As part of the mTORC1 complex, mTOR promotes the phosphorylation of downstream targets including p70S6K and 4EBP1. These events promote ribosome biogenesis and cap-dependent translation, respectively [13, 15, 16].
Rapamycin is a specific inhibitor of mTOR activity within mTORC1. Rapamycin, also an immunosuppressant, complexes with the protein FKBP-12; this complex then binds to mTOR and inhibits its kinase activity . As inhibition of mTOR subsequently inhibits protein translation and cell growth, rapamycin is an excellent candidate for anti-tumor treatment. In fact rapamycin (or similar mTOR inhibitors) has gained interest for the treatment of cancers, including EBV-associated post-transplant lymphoproliferative disease [17–19].
Previous studies have shown that inhibition of mTOR by treatment with rapamycin is effective in inhibiting Kaposi’s sarcoma herpesvirus (KSHV) lytic replication . The inhibition of lytic replication appears to be due to the inhibition of translation of the immediate-early protein, RTA . Another mTOR inhibitor, Torin1, was found to inhibit viral replication for the herpesvirus members cytomegalovirus, herpes simplex virus 1, and murine gammaherpesvirus 68 . These three viruses were much less sensitive to rapamycin treatment . In the case of human cytomegalovirus, the inhibition of mTOR did not greatly affect the immediate-early proteins, as for KSHV, but appeared to inhibit downstream replication events .
In a Drosophila model system, we identified Tor as a modifier of Z and R activities. Translating this finding to the context of lytically-replicating EBV, we found that mTORC1 inhibition via rapamycin treatment yielded different effects in B cell versus epithelial cell lines. While rapamycin treatment of EBV-positive B cells inhibited lytic replication, rapamycin treatment increased lytic replication in the EBV-positive epithelial cell lines tested, suggesting that the effects of mTOR inhibition differ greatly, in respect to lytic replication, between different cell types. These effects upon EBV lytic replication appear to be, at least in part, due to differential influences upon Z and R gene expression.