An EBNA3C-deleted Epstein-Barr virus (EBV) mutant causes B-cell lymphomas with delayed onset in a cord blood-humanized mouse model

Abstract

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.

Latency and lytic replication in Epstein–Barr virus-associated oncogenesis

“…This persistence was associated with EBNA2-driven proliferation during the first month of infection, which then, switched to EBV latency 0 persistence with only non-translated EBER expression after three months 22 . The observed absence of EBV latency III seems to be caused by a combination of EBNA3A or EBNA3C deficiency and immune control of rare completely virus transformed B cells, because in a HIS mouse model with less immunocompetence, LMPexpressing EBNA3C-negative lymphomas can be observed at lower frequency compared to wild-type EBV infection 59 . These findings suggest that EBV persistence might be achieved with minimal or no EBV latency III infection.…”

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

“…In mice reconstituted with functional components of a human immune system it is possible to study EBV's biology in vivo (Münz, 2015), but steps early after infection are difficult to investigate in this tractable model. To our knowledge, an EBNA2-deleted virus has not been studied in this animal model, yet, but, unexpectedly, EBV strains incapable of expressing EBNA3C were found to establish latency and cause B cell lymphomas albeit at a reduced frequency compared with wild-type EBV (Murer et al, 2018;Romero-Masters et al, 2018). EBNA3C is a transcriptional repressor and has been found to abrogate the expression of cell cycle inhibitors such as p16 INK4A encoded by CDKN2A, which is a prerequisite to establish lymphoblastoid cell lines with EBNA3C-deleted EBVs in vitro (Skalska et al, 2013).…”

“…B cells infected with EBNA3C deleted mutant EBVs ceased to proliferate about two weeks post infection(Skalska et al, 2013) very much in contrast to the situation in vivo. In hu-mice EBNA3C deleted viruses established latent infection in B cells while the cells expressed appreciable levels of p16(Romero-Masters et al, 2018). Possibly, there are several reasons that may lead to the observed differences, but the comparison demonstrates the limitations of the experimental models with which we can study the biology of EBV infection in vitro.Some of these limitations also became obvious in our experiments.…”

B cells infected with Type 2 Epstein-Barr virus (EBV) have increased NFATc1/NFATc2 activity and enhanced lytic gene expression in comparison to Type 1 EBV infection

“…Thus, the ability of EBV to transform B cells into long-term LCLs in vitro may not adequately model certain aspects of EBV-associated B-cell lymphomas in humans. We have recently established a humanized mouse model that better models many aspects of the human disease, including a role for lytic infection [4], and the development of EBV-induced lymphomas in the absence of LMP1 or EBNA3C [5,6].…”