Abstract 4794: Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture
Authors: Guidry Joseph T. , Songock William K. , Ma Xiaohui , Nathan Cherie-Ann O. , Bodily Jason M. , Scott Rona S. 
Published: July 2017

An epidemic rise in oropharyngeal squamous cell carcinoma (OSCC) infected with human papillomavirus has been observed over the past decade. The majority of HPV+OSCC arise from the tonsils and base of tongue (BOT), which are lymphoid-rich regions that are known to harbor Epstein-Barr virus (EBV). Indeed, we previously reported HPV/EBV co-infection in 25% of tonsillar and 70% of BOT tumors. HPV is a DNA tumor virus that specifically infects proliferating basal cells in stratified squamous epithelia. HPV replication is facilitated by viral oncoproteins E6 and E7, which create an S-phase-like environment via degradation of cell cycle regulators p53 and Rb, respectively. EBV is also an established DNA tumor virus that replicates in differentiated epithelia in its normal lifecycle.  However, EBV exhibits a latent infection characterized by oncogene expression and lack of viral replication in EBV-associated carcinomas. E6/E7-mediated alteration of the cell cycle results in an interruption in epithelial differentiation, which is required for EBV replication in epithelial cells. Thus, we hypothesize that HPV immortalization of keratinocytes shifts the EBV life cycle from replication to latency.

To test our hypothesis, we developed an EBV/HPV co-infection model utilizing organotypic raft culture to produce stratified epithelia in vitro. Tonsillar keratinocytes immortalized by HPV were EBV-infected by co-culture with IgG-induced EBV-positive Akata Burkitt’s lymphoma cells four days after differentiation induction. Raft tissues were harvested 6 days post-EBV infection for analysis of viral life cycles. EBV infection did not alter HPV replication or gene expression. In HPV-negative tonsillar keratinocytes, robust EBV replication was observed that was blocked by treatment with acyclovir, a nucleoside analog that inhibits EBV lytic replication. In contrast, a dramatic decrease in EBV DNA levels was observed in HPV-positive keratinocytes, which was not further reduced by acyclovir. Human telomerase-immortalized normal oral keratinocytes supported robust EBV replication in organotypic raft culture suggesting a specific effect of HPV rather than immortalization on EBV replication. Expression of E7 alone was sufficient to decrease EBV DNA levels. Preliminary data suggest no change in EBV infectivity between HPV-negative and HPV-positive rafts, and an increase in EBV latency-associated gene expression in HPV-positive cells. The ability of EBV to establish persistent latent infection in HPV-infected epithelial cells would contribute additional viral oncogene expression to explain the rapid development and progression of HPV+OSCC.

Citation Format: Joseph T. Guidry, William K. Songock, Xiaohui Ma, Cherie-Ann O. Nathan, Jason M. Bodily, Rona S. Scott. Reduced Epstein-Barr virus replication in human papillomavirus-immortalized keratinocyte organotypic raft culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2017-4794