Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes

Abstract

Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.

Epstein–Barr virus LMP1 protein promotes proliferation and inhibits differentiation of epithelial cells via activation of YAP and TAZ

“…As uninfected NOKs spontaneously differentiate in monolayer culture when grown at subconfluent conditions without growth factors, and we previously showed that WT-EBV infection inhibits this differentiation ( 14 ), we determined if LMP1 expression is also required for this EBV effect. Immunoblot analyses were performed to compare expression levels of cell cycle markers and markers of epithelial cell differentiation when uninfected, WT-EBV infected, or ∆LMP1-infected NOKs were grown in growth-factor restricted conditions for 24 h. As shown in Fig.…”

“…NOKs provide an excellent model system to study EBV in epithelial cells since they can be stably infected with EBV (in the presence of antibiotic selection) and retain the ability to differentiate ( 4 , 11 – 13 ). Although we recently showed that EBV infection of NOKs inhibits spontaneous differentiation and promotes proliferation when cells are grown in monolayer culture in the absence of supplemental growth factors ( 14 ), suspended in methylcellulose ( 12 ), or “rafted” ( 12 ), the mechanism(s) for these effects and the specific viral protein(s) and/or viral RNAs required have not been previously identified. We show here that mutation of the EBV LMP1 gene greatly reduces the ability of EBV infection to enhance cellular proliferation and inhibit spontaneous differentiation in NOKs when growth factors are limiting.…”

Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity

“…Likewise, HPV16 oncoprotein E6 induces cell proliferation by dephosphorylating a serine residue of YAP1 (S379) and preventing its degradation by the proteasome complex (He et al, 2015). More recently, it has been discovered that Epstein-Barr virus subverts the YAP/TAZ pathway in lytic reactivation in epithelial cells (Singh et al, 2023a; Van Sciver et al, 2021).…”

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice

“…A GFP/G418R cassette was inserted into the non-essential BXLF1 gene of the AG876 type 2 EBV strain genome in the AG876 BL cell line by homologous recombination using G418 selection, and then stably infected HeLa cell clones containing this virus were derived as previously described [ 84 ]. The CRISPR/CAS9 technique was used in the AG876 virus-infected HeLa cells to create a mutation in the EBNA2 gene that puts the reading sequence out-of-frame at EBNA2 residue 25.…”