Changes in the Nasopharyngeal Carcinoma Nuclear Proteome Induced by the EBNA1 Protein of Epstein-Barr Virus Reveal Potential Roles for EBNA1 in Metastasis and Oxidative Stress Responses

Epstein-Barr virus (EBV) infection is causatively associated with a variety of human cancers, including nasopharyngeal carcinoma (NPC). The only viral nuclear protein expressed in NPC is EBNA1, which can alter cellular properties in ways that may promote oncogenesis. Here, we used 2-dimensional difference gel electrophoresis (2-D DiGE) to profile changes in the nuclear proteome that occur after stable expression of EBNA1 in the EBV-negative NPC cell line CNE2. We found that EBNA1 consistently altered the levels of a small percentage of the nuclear proteins. The identification of 19 of these proteins by mass spectrometry revealed that EBNA1 upregulated three proteins affecting metastatic potential (stathmin 1, maspin, and Nm23-H1) and several proteins in the oxidative stress response pathway, including the antioxidants superoxide dismutase 1 (SOD1) and peroxiredoxin 1 (Prx1). Western blot analysis verified that EBNA1 expression upregulated and EBNA1 silencing downregulated these proteins. In addition, transcripts for stathmin 1 were induced by EBNA1, whereas EBNA1 only affected Prx1 and SOD1 at the protein level. Further investigation of the EBNA1 effects on the redox pathway showed that long-term EBNA1 expression in NPC resulted in increased reactive oxygen species (ROS) and increased levels of the NADPH oxidases NOX1 and NOX2, known to generate ROS. In addition, EBNA1 depletion in EBV-positive cells decreased NOX2 and ROS. The results show multiple roles for EBNA1 in the oxidative stress response pathway and suggest mechanisms by which EBNA1 may promote NPC metastases.