Background

EBV is a gammaherpes virus with both latent and lytic infection programs (Young et al., 2016). Latent infection is characteristic of nasopharyngeal carcinoma (NPC), but lytic infection and the production of progeny virus is important for replenishing the local viral reservoir (Tsao et al., 2012; Raab-Traub et al., 2015; Shair et al., 2018). More than 90% of people acquire EBV by adulthood and these chronic carriers continue to shed and transmit EBV in the nasal and oral cavities (Sitki-Green et al., 2003; Hadinoto et al., 2009). However, only a subset of individuals develops EBV-associated NPC, which occurs at high incidence in specific populations, such as South East Asians and Alaskan Innuits (Yu and Yuan, 2002). Both latent and lytic infection are thought to be important in the development of NPC, but this process has been difficult to study because of the rarity of EBV-infected nasopharyngeal cells in asymptomatic carriers, and the lack of physiologically relevant in vitro infection models (Caves et al., 2018; Shair, 2020). To address the question of which epithelial cell types in the nasopharynx are susceptible to EBV infection, and which cell types result in latent vs. lytic infection, we have built two distinct experimental models of the nasopharynx. The EBV infection program depends on epithelial differentiation, which is more accurately modeled in three-dimensional (3-D) culture. Thus, 3-D cell culture models overcome the shortcomings of previous EBV infection studies conducted in 2-D culture, which are limited to studies of EBV latency in outgrowing cell lines.

For the pseudostratified air-liquid interface model (pseudo-ALI model), a pseudostratified epithelium derived from nasopharyngeal conditionally reprogrammed cells (CRC) is exposed to de novo EBV infection by co-culture with anti-human IgG-reactivated EBV-positive B-cell inoculum (Maruo et al., 2001), or by cell-free virus infection. In the presence of Rho-associated protein kinase inhibitors (ROCKi), primary epithelial cells grown with feeder irradiated 3T3-J2 mouse fibroblasts are induced into a highly proliferative state by conditional reprogramming, which allows for propagation of primary cells while maintaining their differentiation potential (Chapman et al., 2010; Liu et al., 2012; Suprynowicz et al., 2012). Subsequently, UNC/USG (University of North Carolina/Ultroser G) specialized media is used to differentiate CRCs into mature pseudostratified airway epithelia in air-liquid interface cultures. Originally developed at the University of North Carolina (Fulcher and Randell, 2013), the UNC component has additives that induce the differentiation of human bronchial epithelial (HBE) and nasopharyngeal epithelial cells, which do not grow well in serum-containing media; thus, it’s necessary to use bovine pituitary extract (BPE) as a serum substitute. Other key constituents of the UNC media include a multitude of growth factors and trace elements that are essential for airway epithelial differentiation (Fulcher and Randell, 2013). These include retinoic acid, which promotes mucocilliary differentiation, and hydrocortisone, which promotes growth and epithelial sodium channel (ENaC) activity. The USG component is needed to promote robust electrophysiology (Gentzsch et al., 2017). The protocol we describe here can be used to differentiate both nasopharyngeal and bronchial epithelial cells (Fulcher and Randell, 2013; Serrano Castillo et al., 2019), but the EBV infection experiments we describe have only been attempted with nasopharyngeal cells.

Akin to EBV infection in organotypic rafts generated from primary oral keratinocytes (Temple et al., 2014, 2017), we generated nasopharyngeal-derived organotypic rafts to study EBV infection in the nasopharyngeal stratified epithelium, using CRCs which were exposed to reactivated EBV by the co-culture method. The principle of organotypic rafts is to generate multilayered stratified epithelia by differentiating keratinocytes at the air-liquid interface (Kopan et al., 1987) in defined E-media, which was originally described for the differentiation of oral (Temple et al., 2017) and cervical epithelial cells (Meyers, 1996). Here, we generated 3-D cell cultures using nasopharyngeal cells from donors undergoing skull base surgery (without any upper airway pathology), and from patients undergoing sinus surgery. CRCs can also be derived from the inferior turbinate from healthy persons without any co-pathology, provided such donors with no reason for surgery can be recruited. In sufficient quantity, the CRCs can be cryopreserved and thawed to generate both types of 3-D cell culture. A major advantage of using cryobanked cells is reproducibility and assessment of donor variability. These 3-D culture methods, when combined with EBV infection, are expected to reveal susceptible cell types in the upper airway, and uncover host determinants of EBV (latent and lytic) infection. A schematic of the workflow and measurable experimental outcomes are described in the Diagram below.

Part I: Differentiation into Pseudostratified-ALI Culture