Dynamics of Epstein-Barr virus episomes in infected epithelial cells

Abstract

Epstein-Barr virus (EBV) is the first identified human tumor virus. It readily infects human memory B cells and establishes life-long latency after primary infection. EBV infection is also associated with specific types of carcinomas with lymphoepithelioma properties, including the undifferentiated nasopharyngeal carcinoma and EBV-associated gastric adenocarcinoma. EBV genomes are found in almost all of these cancer cells. However, how the EBV genomes persist in the proliferating host cells is poorly understood. The replication of EBV genomes in each cell cycle and their partitioning to daughter cells are believed to be crucial for the persistence of EBV in host cells. Previous studies have either investigated the replication and partition of EBV in live cells using EBV-derived plasmids which contained partial EBV genome or through detection of EBV episomes by FISH in fixed cells. The replication and partition of the whole EBV episomes in live cells have never been examined.

In the present study, the dynamics of the replication and segregation of the whole EBV episomes during latent infection in live epithelial cells was examined for the first time. A newly engineered visible EBV was used in the study. The dynamics of replication and partitioning of visible EBV genomes were characterized at single cell level. Interestingly, unlicensed replication of EBV genomes (EBV replicated more than once per cell cyle) was observed for the first time in 13.6% of the infected cells. Licensed replication of EBV (EBV replicated once per cell cycle) was observed in 63.6% of cells, and no-replication of EBV (no EBV replication per cell cycle) was observed in 22.7% of cells. On average, EBV genomes replicated to 1.61 folds per cell cycle per cell. In addition, the EBV episomes preferred to localize at host nuclear regions active for replication and transcriptions. Lastly, EBV episomes from mother cells partitioned unequally into daughter cells. The unlicensed replication and unequal partitioning of EBV episomes may compensate for the imperfect replication, giving rise to a wide range of distribution of EBV copy per cell within a clonal population.

The present study characterized the dynamics of EBV replication and partitioning in epithelial cells during latent infection at single cell level. This study also contributed to the mechanism for EBV maintenance in epithelial cells. The establishment of latent infection of visible EBV in epithelial cells and the imaging-analysis system developed in this study can be applied to future investigation of virus-host interaction to further our understanding of the role of EBV in the pathogenesis of the EBV-associated carcinoma.