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. 2022 Oct 3;18(10):e1010868.
doi: 10.1371/journal.ppat.1010868. eCollection 2022 Oct.

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

Deo R Singh  1 , Scott E Nelson  1 , Abigail S Pawelski  1 , Juan A Cantres-Velez  1 , Alisha S Kansra  1 , Nicholas P Pauly  1 , Jillian A Bristol  1 , Mitchell Hayes  1 , Makoto Ohashi  1 , Alejandro Casco  1 , Denis Lee  1 , Stuart A Fogarty  1 , Paul F Lambert  1 , Eric C Johannsen  1   2 , Shannon C Kenney  1   2
Affiliations

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

Deo R Singh et al. PLoS Pathog. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Gene set enrichment analysis (GSEA) suggests increased proliferation, decreased keratinocyte differentiation and decreased E-cadherin signaling in both the type 1 EBV-infected and type 2 EBV-infected NOKs in comparison to uninfected NOKs when growth factors are limiting.
AG876 virus-infected, Akata virus-infected, or uninfected NOKs were grown in growth factor-restricted conditions, and RNA-seq and GSEA were performed as described in the Materials and Methods sections. Displayed are GSEA results on a focused set of differentially expressed pathways related to EBV-infected versus uninfected NOKs following interrogation of extensive GSEA results shown in S4 Fig. Displayed pathways contain a Benjamini-Hocheberg (BH)-adjusted p-value of <0.05 and are sorted by Normalized Enrichment Scores (NES). Pathways upregulated in the EBV-infected NOKs relative to uninfected NOKs are associated with NES values greater than 0, and down-regulated pathways are associated with NES values less than 0.
Fig 2
Fig 2. Both type 1 and type 2 EBV-infected NOKs proliferate faster than uninfected NOKs when growth factors are limiting.
50,000 uninfected NOKs, Type 1 Akata EBV-infected NOKs, or Type 2 AG876 EBV-infected NOKs (each in the context of the “NOKs-2” line) were uniformly seeded in each well of a 6 well plate, and then grown in KSFM medium without any EGF or BPE supplement. After 5 days, cells were counted using trypan blue staining. The total cells obtained from each Akata EBV-infected or AG876 EBV-infected NOKs condition was normalized to the number of cells obtained from uninfected NOKs conditions. The normalized data was plotted as a bar chart. Individual data points represent the fold- growth increased relative to uninfected NOKs (set as 1). The bars represent the average value of fold-increase relative to uninfected NOKs. The error bars represent the standard error. Statistical analysis was performed using one-way ANOVA. p<0.05 was considered statistically significant.
Fig 3
Fig 3. Type 1 Akata virus and type 2 AG876 virus both induce proliferation in NOKs when growth factors are limiting.
Uninfected, Akata EBV-infected, or AG876 EBV-infected NOKs (each in the context of the “NOKs 2” line) were seeded in triplicate (125K cells per well) in 6 well plates and grown in KSFM medium without supplements for 24 hours. Immunoblot analysis was then performed to assess expression levels of LMP1, PCNA, p21, or HSP90 (loading control) as indicated.
Fig 4
Fig 4. Type 1 and Type 2 EBV both induce EMT in NOKs.
Uninfected, Akata EBV-infected, or AG876 EBV-infected NOKs (each in the context of the “NOKs-2 line”) were seeded in triplicate (125K cells per well) in 6 well plates and grown in KSFM medium without supplements for 24 hours. Immunoblot analysis was then performed to assess expression levels of E-cadherin, Vimentin, Fibronectin or tubulin (loading control).
Fig 5
Fig 5. Type 1 and Type 2 EBV both induce NF-κB signaling in NOKs.
Uninfected, Akata EBV-infected, or AG876 EBV-infected NOKs (each in the context of the “NOKs-2” line) were seeded in triplicate (125K cells per well) in 6 well plates and grown in KSFM medium without supplements for 24 hours. Immunoblot analysis was then performed to assess expression levels of p100/p52, total p65, phospho-p65, or tubulin (loading control). The same cellular extracts were used in this figure as in Fig 4.
Fig 6
Fig 6. Type 1 and Type 2 EBV both inhibit spontaneous NOKs differentiation when growth factors are limiting.
Uninfected, Akata EBV-infected, or AG876 EBV-infected NOKs (each in the context of the “NOKs-2 line”) were seeded in triplicate (125K cells per well) in 6 well plates and grown in KSFM medium without supplements for 24 hours. Immunoblot analysis was then performed to assess expression levels of Keratin-10 (K-10), Involucrin, ZNF750, BLIMP1, KLF4, delta p63, IRF6, TGM1 or tubulin (loading control) as indicated.
Fig 7
Fig 7. Type 1 and Type 2 EBV both inhibit methylcellulose-induced NOKs differentiation.
Uninfected, Akata EBV-infected, or AG876 EBV-infected NOKs (each in the context of the “NOKs-2” line) were differentiated in 1.6% methylcellulose containing KSFM medium without supplements for 24 hours. Following differentiation, the cells were harvested and immunoblot analysis was then performed to assess expression levels of LMP1, K-10, Involucrin, GHRL3, ZNF750, KLF4, TGM1, SPRR1A and tubulin (loading control) as indicated.
Fig 8
Fig 8. Type 1 and type 2 EBV both inhibit NOKs differentiation during rafting.
Two conditions each of uninfected NOKs, Akata EBV-infected NOKs, and AG876 EBV-infected NOKs (each in the context of the “NOKs 2” line) were differentiated on raft cultures as described in the methods. Histology was performed on rafted cells and sections were stained for hematoxylin and eosin (H&E) or stained with anti-K-10 antibody and counterstained with hematoxylin. Representative sections are shown for uninfected NOKs and NOKs infected with Akata EBV or AG876 EBV.
Fig 9
Fig 9. NOKs infected with Type 1 and type 2 EBV exhibit similar levels of lytic reactivation in response to differentiation stimuli.
(A) Uninfected NOKs, two different lines of AG876 EBV-infected NOKs cells, and four different lines of Akata EBV-infected NOKs (each in the context of the “NOKs-2” line) were grown in sub-confluent conditions in KSFM medium without supplements for 24 hours. Cells were then treated with TPA at 20ng/mL for 48 hours, and immunoblot analysis was performed to assess expression of BZLF1, BRLF1, LMP1, BMRF1 or tubulin (loading control) as indicated. (B) Akata EBV-infected, AG876-EBV infected or uninfected NOKs were differentiated by suspending the cells in 1.6% methylcellulose containing KSFM medium without supplements for 72 hours, and then immunoblot analysis was performed to assess expression of the BZLF1, BRLF1, LMP1, BMRF1 and tubulin (loading control) proteins.
Fig 10
Fig 10. Rafted Type 1 and Type 2 EBV infected NOKs have similar levels of lytic EBV reactivation.
Two lines each of Akata EBV-infected or AG876 EBV-infected cells were differentiated on raft cultures (each in the context of the “NOKS-2 line”). (A) Histology was performed on rafted cells and sections were stained with BZLF1 antibody and counter-stained with hematoxylin (choosing regions of the raft that had some level of K10 expression). Representative sections are shown for NOKs infected with Akata EBV or AG876 EBV. BZLF1-expressing cells are indicated by arrows. (B) In Situ hybridization was performed to detect EBV EBERs. Representative sections are shown for uninfected NOKs or NOKs infected with AG876 EBV.
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