Date Published: August 20, 2018
Publisher: Public Library of Science
Author(s): James C. Romero-Masters, Makoto Ohashi, Reza Djavadian, Mark R. Eichelberg, Mitch Hayes, Jillian A. Bristol, Shidong Ma, Erik A. Ranheim, Jenny Gumperz, Eric C. Johannsen, Shannon C. Kenney, Paul D. Ling.
EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.
Epstein-Barr virus (EBV) is a human gamma-herpesvirus that infects 90% of the world’s adult population . EBV establishes a lifelong infection in the memory B cell compartment, and periodically reactivates to the lytic form of viral infection when B cells are stimulated by antigen and/or differentiate into plasma cells [1–3]. In addition to causing the clinical syndrome, infectious mononucleosis, EBV is associated with multiple human malignancies of B-cell and epithelial origin, including Burkitt lymphoma, Hodgkin lymphoma, diffuse large B cell lymphoma (DLBCL), post-transplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma and gastric carcinoma [4–6]. All EBV-associated malignancies are latently infected with EBV.
EBNA3C is expressed in human B-cell lymphomas that have type III latency, and in a subset of human Burkitt lymphomas [6,73]. The EBV EBNA3C protein plays an essential role in promoting EBV transformation of B cells into LCLs in vitro, and this effect is largely due to the ability of EBNA3C (in collaboration with EBNA3A) to inhibit expression of the CDKN2A tumor suppressor locus. Nevertheless, most EBV-infected lymphomas in humans have restrictive forms of viral latency that do not express EBNA3C , indicating that EBV-infected lymphoma cells in vivo can sometimes proliferate even without EBNA3C. Furthermore, although EBNA3C regulates numerous different cellular genes in addition to CDKN2A, whether these other EBNA3C-regulated cellular genes have important in vivo functions remains unclear.
While this manuscript was being revised, another group reported that an EBNA3C-deleted virus is able to establish latency in another humanized mouse model .