Maintenance of Epstein-Barr virus latent genome in epithelial tumor cells during the cellular clonal expansion / 中南大学学报(医学版)

Objective To determine the maintenance and loss of Epstein-Barr virus (EBV) genome during the clonal expansion of the EBV-infected epithelial cells. Methods The epithelial tumor cell line, 293-EBV, in which the EBV genome was observed with green fluorescent protein (GFP) readout. After a dozen of passages, it contained cells with strong or weak GFP expression, and some with complete loss of EBV genome. The cell growth was then continuously observed under a confocal microscope. The cell dividing and GFP expression were also observed during the clonal expansion by being made into very low density. Results The cells moved around due to adherence and mobility, while the GFP expression remained unchanged in the undivided cells. The cells could form compact or loosen clones. The EBV genome easily persisted in those clones when cells were growing compactly. As the cell number increased, the GFP expression became weak or even died away at the sites of low density in the loosen clones. Conclusion EBV-positive epithelial cells are able to sustain the EBV genome during its clonal expansion. The cells maintain EBV genomes by passing them to the daughter cells after replication. When the cells unsuccessfully inherit the EBV genome, the daughter cells may lose them which is related to the low cell density as well as the epithelial environment.

Infectious Cloning Approach for Herpesvirus Based on Bacterial Artificial Chromosomes / 中国生物工程杂志

The genetic analysis of herpesviruses has been a constant challenge, due to the large, complex genomes of herpesviruses and mutagenesis of viral genes by conventional recombination methods in cell culture. Recently, a completely new approach for full-length infectious clones of herpesviruses based on bacterial artificial chromosomes (BACs) has been developed. This technique allows the maintenance, propagation and genetic modification of the viral genome as a BAC plasmid in E.coli, thus making the procedures fast, safe and effective in prokaryotic cells. This technique also makes it possible for the reconstitution of viral progeny or mutants by transfection of the BAC plasmid into eukaryotic cells, thereby facilitating the analysis of viral gene functions in the context of genome. In this presentation, Epstein-Barr virus was used as an example to describe the principle, establishment of the technique and mutation introduction into the BAC plasmid, and to discuss the perspective in the use of BAC-cloned herpesviruses.