| Epstein-Barr virus (EBV) is a γ-herpesvirus associated with several malignancies. Although the virus encodes more than eighty proteins, very few are expressed in these tumors. However, lytic viral gene expression offers a multitude of enzymatic and other targets that may facilitate tumor-specific therapeutic approaches. Bortezomib, a proteasome inhibitor, is a potent inducer of EBV lytic gene expression. The mechanism of bortezomib's activity is not completely understood, but the induction of ER stress and the unfolded protein response (UPR) may be involved. We wanted to determine how bortezomib induces the EBV lytic cycle. Treating EBV-positive Burkitt lymphoma cells with bortezomib induces the transcription factor, CCAAT/enhancer binding protein (C/EBPβ). We created a C/EBPβ knockdown cell line and show C/EBPβ is required for bortezomib-mediated lytic induction. Further analysis of RNA and protein reveal induction of C/EBPβ occurs in the context of ER stress and UPR activation. Suppression of UPR signaling, by cycloheximide treatment or BiP (a UPR sensor) knockdown inhibits bortezomib-mediated EBV lytic induction, but not bortezomib-mediated cytotoxicity. Other ER stress-inducing agents similarly activate the EBV lytic cycle in EBV-positive lymphoid and epithelial cell lines, as well as in Kaposi sarcoma herpes virus (KSHV)-positive primary effusion cell lines. The HIV protease inhibitor, nelfinavir, has been shown to induce ER stress and the UPR. Treatment with nelfinavir induces EBV lytic gene expression in Burkitt lymphoma cells. Inhibiting UPR signaling hinders nelfinavirmediated EBV lytic reactivation, but not nelfinavir-mediated cytotoxicity. Although nelfinavir stimulates EBV replication, nelfinavir inhibits virion production. We conclude that bortezomib activates the EBV lytic cycle through induction of C/EBPβ and the UPR. Furthermore, any agent that induces the UPR in EBV- or KSHV-positive cell lines can induce lytic reactivation. We identify Nelfinavir as a novel inducer of the EBV lytic cycle and inhibitor of virion production. Finally, Bortezomib and nelfinavir's cytotoxic effects are separable from their viral effects. These drugs may serve as potential therapeutic agents for EBV-associated malignancies. |
