A study of the epstein barr virus nuclear antigens EBNA3A and EBNA3C in vitro and in vivo

Abstract

The lymphotropic gamma-herpesvirus Epstein Barr virus (EBV) is ubiquitous within the human population, but it is etiologically associated with several B cell malignancies. In vivo, EBV infects and activates naïve B cells to differentiate into memory B cells (MBC), where viral persistence is established for the lifetime of the host. The oncogenic EBV nuclear antigens EBNA3A and EBNA3C are transcriptional regulators required for EBV-mediated oncogenic progression and cell cycle deregulation. This study encompasses in vitro and in vivo approaches to further investigate the roles of EBNA3A and EBNA3C. To further characterise the independent and overlapping functions of EBNA3A and EBNA3C, bacterial artificial chromosome (BAC) and estrogen receptor (ER) fusion technology were used to generate a recombinant EBV where EBNA3A and EBNA3C were both conditionally expressed. Proliferation and viability analysis of primary B cells infected with this virus in vitro revealed the absence of EBNA3A and EBNA3C is cumulatively detrimental to cells, and activation of cell cycle regulatory factors was more profound. Protein and qPCR analysis of EBV-infected cells lacking both EBNA3A and EBNA3C also revealed elevated levels of the plasma cell (PC) differentiation factors p18INK4c and BLIMP-1 at 20 days post infection. Consistent with increased PC differentiation factors, flow cytometric analysis showed EBNA3A/EBNA3C-null cells had a PC-like phenotype, with increased CD138 and CD38 cell surface antigens, alongside elevated immunoglobulin production. This suggests that alongside their oncogenic functions, EBNA3A and EBNA3C have evolved to suppress the B cell to PC differentiation pathway after infection with EBV. This likely favours the alternative MBC differentiation pathway, and therefore long-term EBV latency and persistence. To assess the roles of EBNA3A and EBNA3C in vivo, a mouse model with human immune system components was established. Humanised mice were infected with EBV lacking the EBNA3A and EBNA3C genes. Immunohistochemistry and qPCR for viral factors demonstrated EBV can persist in the absence of EBNA3A and EBNA3C expression for 6 weeks, and viral pathology was seen. The viability of using conditional ER-fusion recombinant EBV viruses in vivo was also assessed.