Functional characterization of Epstein-Barr virus-encoded RNA 2 in the pathogenesis of nasopharyngeal carcinoma

Abstract

Epstein-Barr virus (EBV)-encoded RNA 2 (EBER2) promotes B cell transformation in primary B cell and lytic replication of EBV in Burkitt lymphoma and lymphoblastoid cell lines. EBER2 recruits transcription factors such as Pax5 and PU.1 to mediate changes in gene transcription to regulate the latency of EBV and proliferation in B cells. In contrast, the pathogenic mechanism of EBER2 in nasopharyngeal carcinoma (NPC) is largely unknown. High risk EBV variants containing 4-bp deletion and single nucleotide polymorphisms within EBER2 (designated as NPC-EBV) when compared to wild type EBV (wt-EBV) are found to be highly associated with Southern Chinese NPC. Here, I set out to (i) investigate the structures and functions of EBER2 and (ii) compare the EBER2 functional properties of NPC-EBV and wt-EBV in NPC.

The structure of EBER2 was first determined by an experimental assay known as selective 2′-hydroxylacylation analyzed by primer extension and mutational profiling (SHAPE-MaP). SHAPE-MaP revealed two arms forming stem loop structures at the 5′-end (5′-arm) and 3′-end (3′-arm), respectively, which may represent the accessible regions of EBER2 for the binding of either RNA or protein. EBER2 of NPC-EBV (represented by M81 variant) and that of wt-EBV (represented by B95-8) share similar structures but differ in the 5′-arm. I postulated that specific cellular proteins may bind directly at the 5′- or 3′-arm of EBER2. Using RNA antisense precipitation (RAP) and mass spectrometry, I identified two novel EBER2-interacting proteins, namely, N(6)-methyladenosine (m6A) methyltransferase (METTL16) and spliceosome-associated factor 3 (SART3) in EBV-positive NPC cell line, C666-1. Furthermore, the C-terminal vertebrate conserved region (VCR) of METTL16 and RNA recognition motifs (RRMs) of SART3 were found to be the main binding domains of the respective proteins to EBER2. Next, the 5′- and 3′-arms of EBER2 were systematically truncated to locate the corresponding binding sites on EBER2. Whereas the interaction of EBER2 with either METTL16 or SART3 was not altered by deletions at the 5′-arm, a slight decrease of the level of EBER2 with deletions at the 3′-arm and a marked decrease of the level of EBER2 with deletions at both 5′- and 3′-arms were observed.

Overexpression of M81 and B95-8 EBER2 variants increased the proliferation rate of C17 cell line whilst silencing of EBER2 significantly decreased the proliferation rate of C666-1 cell line. Both EBER2 variants also promoted cell migration, cell cycle progression and lytic replication of EBV and downregulated IFN-stimulating genes such as Interferon-alpha inducible protein 6 (IFI6) in C17 cell line. Overexpression of M81 EBER2 decreased the m6A level of cyclin D1 mRNA and mediated translocation of SART3 from cytosol to nucleus.

In conclusion, two novel proteins, METTL16 and SART3, are identified to bind directly to the 5′ and 3′ arms of EBER2 through specific domains of the proteins in NPC. EBER2 promotes cell proliferation and migration, cell cycle progression and modulates IFN-stimulating gene expression and m6A level of target mRNA in NPC. Despite different secondary structures of EBER2 in NPC-EBV and wt-EBV, their ability to bind METTL16 and SART3 and their cellular consequences in NPC are similar.