Calcium mobilization is responsible for Thapsigargin induced Epstein Barr virus lytic reactivation in in vitro immortalized lymphoblastoid cell lines

The latent state is a critical component of all herpesvirus infections, and its regulation remains one of the most active areas of Epstein-Barr Virus (EBV) research. In particular, identifying environmental factors that trigger EBV reactivation into a virus-productive state has become a central goal in EBV latency research. Recently, a category of chemicals known as inducers of the endoplasmic reticulum unfolded protein response (UPR) have been shown to trigger EBV lytic reactivation in various established EBV-associated lymphoma cell lines. This has led to the recent belief that UPR is a universal cellular signaling pathway that directly triggers EBV lytic reactivation irrespective of cell type. We tested the potency of several widely used UPR inducers for EBV lytic reactivation on virus-immortalized primary lymphoblastoid cell lines (LCLs) in vitro. We found that, with the exception of Thapsigargin (Tg), UPR inducers did not trigger significant increases in BZLF1 transcripts or changes in the numbers of EBV genomic copies/cell in our panel of primary LCLs. Further investigation revealed that induction of lytic reactivation by Tg appeared to be due to its ability to trigger intracellular Ca(2+) mobilization rather than its ability to induce UPR, based on our observations in which UPR induction alone was not sufficient to trigger the EBV lytic cycle in our LCLs. EBV immortalized LCLs have rarely been included in the majority of the lytic reactivation studies yet the characteristics of latent infection in LCLs should resemble those of proliferating B cells in clinically encountered lymphoproliferative diseases. Based on these observations, we propose an alternative mechanism of action for Tg in triggering EBV lytic reactivation in LCLs, and suggest that the proposed use of any chemical inducers of UPR for a purpose of oncolytic/lytic induction therapy needs to be fully evaluated pre-clinically in a panel of LCLs.