Epstein-Barr Virus Infection Promotes Epithelial Cell Growth by Attenuating Differentiation-Dependent Exit from the Cell Cycle

Abstract

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated cancers, progress in examining EBV’s role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection in vivo, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers. IMPORTANCE Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.

Epstein–Barr Virus Infection of Oral Squamous Cells

“…EMT contributes to cancer progression and metastasis by inducing migration, invasion, and inhibition of cell adhesion [27]. In the present study, EBV infection induced mesenchymal phenotypes, migration, and invasion of infected cells (Figures 6 and 7).…”

“…There are many reports that EBV is involved in the development of oropharyngeal cancer. An immortalized keratinocyte-based model has been shown to be susceptible to EBV [26,27]. Here we show for the first time that EBV establishes a latent infection in two squamous epithelial cells, which were well differentiated HSC1 cells and poorly differentiated SCC25 cells.…”

Epstein-Barr Virus Mediated Signaling in Nasopharyngeal Carcinoma Carcinogenesis

“…Lymphoblastoid cell lines (LCL) represent a good tool to study the formation of EBV-associated lymphomas. However, EBV infection to primary epithelial cells induces lytic infection [ 63 , 64 ], therefore primary epithelial cells are not suitable for oncogenic study. Instead, cell lines originated from cancers are used, because these cell lines often show latent infection of EBV.…”

Epstein–Barr Virus—Oral Bacterial Link in the Development of Oral Squamous Cell Carcinoma

“…EBV has been linked to the development of epithelial cancers such as nasopharyngeal carcinoma and gastric cancer [ 39 , 52 ]. Even though EBV infection in epithelial cells is generally lytic, it has been observed that a latency state can be established in cells that show prior genomic DNA damage, like cyclin D1 overexpression [ 53 , 54 , 55 ].…”