Epstein-Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity

Simple Summary Nasopharyngeal carcinoma is associated with Epstein-Barr virus (EBV) infection and originates junction of the oropharynx and nasal cavity, where stratified squamous epithelium and respiratory epithelium are the lining. To elucidate the mechanisms by which EBV transforms the nasopharyngeal epithelium, a pseudostratified multiple-layer model with cilia forming on the apical surface by air-liquid interface (ALI) culture of primary nasopharyngeal epithelial cells was established. We showed: (1) ALI cultures formed stratified epithelia and maintained the diversity of cells found in the airway epithelium, such as ciliated, muco-secretory, and basal cells. (2) Polarized stratified epithelium was more susceptible to EBV infection than monolayer cells. (3) EBV infection in ALI cultures was verified by showing EBV-encoded RNA expressions. (4) EBV infection disrupted the integrity of the epithelium. Thus, our model can be used not only to examine the pathogenesis of pre-neoplastic EBV-infected cells, but also to develop anti-EBV therapy or early stage NPC treatment. Epstein-Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.