Regulation of lytic Epstein-Barr virus replication in a newly established nasopharyngeal cancer cell line

Abstract

Nasopharyngeal carcinoma (NPC), with a remarkably distinctive ethnic and geographic distribution, has the highest incidence among Southern Chinese. Unlike other types of head and neck cancers, a unique feature of NPC is its close association with Epstein-Barr virus (EBV). Latent EBV infection is found in every cancer cell of virtually all cases of undifferentiated NPC in endemic regions. However, the exact roles of EBV infection during the pathogenesis of NPC remain largely undefined, partially due to the limited in vitro NPC cell models. We recently established a new NPC cell line, NPC43, from a recurrent nasopharyngectomized NPC tissue, and characterized it with positive EBV infection. NPC43 is regularly cultured in medium supplemented with Rho kinase inhibitor, Y-27632, which has been reported with suppressive effect in the differentiation of squamous epithelial cells. For early passages of NPC43 (at P12 and P37), removal of Y-27632 from culture medium leads to enhanced transcription of EBV lytic genes, including BZLF1, BRLF1, BMRF1 and BLLF1, indicated by RT-qPCR results. However, after continuous in vitro culture to late passage (p141), NPC43 exhibits insensitive growth response to the deprivation of Y-27632, while EBV lytic transcripts remains at the comparable levels as those in Y-27632-supplemented culture medium. De novo assembly and phylogenetic analysis of the whole EBV genome in NPC43 based on the reads from whole genome sequencing (WGS) mapped to the reference EBV genome reveals that NPC43-EBV shows significant similarity with M81 and other Hong Kong NPC-EBV genome sequences. Since NPC43 could be induced to complete EBV lytic replication with production of infectious EBV virions by lytic stimuli, including TPA treatment and overexpression of BZLF1, it could serve as a useful model to study the biological features of EBV in nasopharyngeal epithelial cells, as well as the cloning of full-length EBV genomes by CRISPR/Cas9-mediated homology-directed repair.