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

Abstract

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. E pstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is harbored in at least 90% of the world population (40). Transmitted through saliva, EBV infects B lymphocytes as well as the epithelium of the orthopharynx. Upon primary infection, EBV immediately establishes latent infection in cells by maintaining multiple copies of its genome in the form of episomes. While most infected individuals control the virus efficiently and remain free of EBV-associated diseases, latent EBV infection is linked to the development of both lymphoid and epithelial tumors. Nasopharyngeal carcinoma (NPC) is one such epithelial tumor in which EBV is widely recognized as a causative agent, since most NPC tumors are monoclonal proliferations of latently EBV-infected cells (38). These tumor cells express only one EBV nuclear protein, EBNA1, in addition to the EBV latent membrane proteins LMP1 and LMP2 (38).Epstein-Barr nuclear antigen 1 (EBNA1) is essential for the establishment of latent EBV infection since it replicates and segregates the viral genome in proliferating cells (reviewed in reference 17). Additionally, it is a transcriptional activator for other EBV latency genes. Aside from its roles in viral persistence, increasing numbers of observations suggest that EBNA1 alters cellular processes in ways that contribute to host cell survival and proliferation. First, EBNA1 is expressed in all latency forms in proliferating cells and is the only EBV protein...

Ebna1

“…In addition, EBNA1 was found to increase the expression of the NOX2 NADPH oxidase which might account for the ROS induction (Gruhne et al 2009). Similarly, a comparison of the nuclear proteome in NPC cells with and without EBNA expression showed that EBNA1 increased the levels of several oxidative stress response proteins including the antioxidants superoxide dismutase 1 and peroxiredoxin 1, known to be induced by ROS (Cao et al 2011). Further studies confirmed that, in the presence of EBNA1, ROS levels were elevated and that NOX1 and NOX2 transcripts were increased (Cao et al 2011).…”

“…Similarly, a comparison of the nuclear proteome in NPC cells with and without EBNA expression showed that EBNA1 increased the levels of several oxidative stress response proteins including the antioxidants superoxide dismutase 1 and peroxiredoxin 1, known to be induced by ROS (Cao et al 2011). Further studies confirmed that, in the presence of EBNA1, ROS levels were elevated and that NOX1 and NOX2 transcripts were increased (Cao et al 2011). Therefore, EBNA1 appears to have multiple effects on the oxidative stress response, although the mechanisms of these effects are not yet known.…”

Reactive Oxygen Species Are Induced by Kaposi's Sarcoma-Associated Herpesvirus Early during Primary Infection of Endothelial Cells To Promote Virus Entry

“…ROS production is dependent on paracrine secretion of interleukin 10 (IL-10) in latency I, whereas expression of EBV nuclear antigen 2 (EBNA-2) and latent membrane protein 1 (LMP1) is proposed in latency III (66). EBNA-1 could also be involved in ROS production (67,68 carcinoma resulted in an increase in ROS production (69). Taken together, these studies indicated that ROS production induced by EBV could play a role in genome instability as well as in metastasis.…”

Epstein-Barr Virus EBNA1 Protein Regulates Viral Latency through Effects on let-7 MicroRNA and Dicer

“…For example, EBNA1 counter-acts the stabilization of p53 by USP7 and induces the loss of PML nuclear bodies through the degradation of PML proteins, both of which contribute to the ability of EBNA1 to interfere with apoptosis and DNA repair (20,21). EBNA1 has also been found to inhibit transforming growth factor ␤ and NF-B signaling and to induce oxidative stress (22)(23)(24)(25). EBNA1 may also be able to transactivate the expression of some cellular genes.…”