Pharmacologic Activation of Lytic Epstein-Barr Virus Gene Expression without Virion Production

Abstract

Several therapeutic strategies targeting Epstein-Barr virus (EBV)-associated tumors involve upregulation of viral lytic gene expression. Evidence has been presented that the unfolded protein response (UPR) leads to EBV lytic gene expression. Clofoctol, an antibacterial antibiotic, has been reported to upregulate the UPR in prostate cancer cell lines and to slow their growth. We investigated the effects of clofoctol on an EBV-positive Burkitt lymphoma cell line and confirmed the upregulation of all three branches of the UPR and activation of EBV lytic gene expression. While immediate early, early, and late EBV RNAs were all upregulated, immediate early and early viral proteins but not late viral proteins were expressed. Furthermore, infectious virions were not produced. The use of clofoctol in combination with a protein kinase R-like endoplasmic reticulum kinase inhibitor led to expression of late viral proteins. The effects of clofoctol on EBV lytic protein upregulation were not limited to lymphoid tumor cell lines but also occurred in naturally infected epithelial gastric cancer and nasopharyngeal cancer cell lines. An agent that upregulates lytic viral protein expression but that does not lead to the production of infectious virions may have particular value for lytic induction strategies in the clinical setting. IMPORTANCE Epstein-Barr virus is associated with many different cancers. In these cancers the viral genome is predominantly latent; i.e., most viral genes are not expressed, most viral proteins are not synthesized, and new virions are not produced. Some strategies for treating these cancers involve activation of lytic viral gene expression. We identify an antibacterial antibiotic, clofoctol, that is an activator of EBV lytic RNA and protein expression but that does not lead to virion production.

Arsenicals, the Integrated Stress Response, and Epstein–Barr Virus Lytic Gene Expression

“…We have used GFP expression by the BX1 cell line as an indicator of lytic gene expression in this and several previous investigations [ 12 , 26 ]. GFP expression is appreciated in only a small percentage of cells under basal conditions.…”

“…In recent work, we reported that eIF2α phosphorylation, by the protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), activated EBV lytic viral transcription [ 12 ]. Phosphorylation of eIF2α has been extensively studied in other contexts [ 13 ].…”

“…GCN2 is activated by binding uncharged tRNAs that are associated with amino acid starvation. With the observation that PERK-mediated phosphorylation of eIF2α led to activation of EBV lytic gene expression [ 12 ], we were interested in the possibility that the other arms of the ISR might also mediate activation of lytic gene expression, including HRI, which is activated by As 2 O 3 ( Figure 1 A) [ 14 , 15 , 16 ].…”

Lytic Induction Therapy against Epstein–Barr Virus-Associated Malignancies: Past, Present, and Future

“…Proteasome inhibitor such as bortezomib and endoplasmic reticulum (ER) stress inducers such as thapsigargin and tunicamycin, on the other hand, can induce EBV lytic cycle by activating ER stress/unfolded protein response (UPR), which induces JNK and/or C/EBP-β and activates Zp through C/EBP-binding sites in ZII and ZIIIB elements [ 24 , 25 ]. UPR-induced lytic reactivation was also observed in clofoctol treatment, which mediates the activation of the PERK-XBP1 axis [ 26 ].…”

“…Indeed, a pilot study on the efficacy and safety of romidepsin in treating extranodal natural killer/T-cell lymphoma found a substantial increase in viremia in these patients [ 52 ]. The novel compound C7 [ 36 , 42 , 46 ] and anti-bacterial antibiotic, clofoctol [ 26 ], were found to induce the expression of immediately early and early lytic proteins, but not late lytic proteins. Moreover, EBV virions were not produced after lytic induction by these two compounds.…”