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Epstein-Barr virus (EBV) is an important human dsDNA virus, which has been shown to be associated with several malignancies including about 10% of gastric carcinomas. How EBV enters an epithelial cell has been an interesting project for investigation. “Cell-in-cell” infection was recently reported an efficient way for the entry of EBV into nasopharynx epithelial cells. The present approach was to explore the feasibility of this mode for EBV infection in gastric epithelial cells and the dynamic change of host inflammatory reaction. The EBV-positive lymphoblastic cells of Akata containing a GFP tag in the viral genome were co-cultured with the gastric epithelial cells (GES-1). The infection situation was observed under fluorescence and electron microscopies. Real-time quantitative PCR and Western-blotting assay were employed to detect the expression of a few specific cytokines and inflammatory factors. The results demonstrated that EBV could get into gastric epithelial cells by “cell-in-cell” infection but not fully successful due to the host fighting. IL-1β, IL-6 and IL-8 played prominent roles in the cellular response to the infection. The activation of NF-κB and HSP70 was also required for the host antiviral response. The results imply that the gastric epithelial cells could powerfully resist the virus invader via cell-in-cell at the early stage through inflammatory and innate immune responses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-019-00097-1) contains supplementary material, which is available to authorized users.

A tumor model that Epstein-Barr virus (EBV) latent infection facilitated the tumorigenicity was previously established using the Maxi-EBV system. In the present approach, EBV-lost cell clones demonstrated significantly decreased tumorigenesis. On the other hand, the LMP1 gene in Maxi-EBV genome was replaced by that of nasopharyngeal carcinoma origin. The resultant cell line, 293–1/NL showed much lower malignancy than the original 293-EBV. The result was opposite to our expectation. The change of 293 sublineage cells for EBV harboring also got similar result. To seek the underlying reason, the copy number of EBV genome in all the cell lines was detected. The result indicated that 293-EBV contained about 4.5-fold higher EBV copies than 293–1/NL did. Parallel EBV genomes led to relatively stable copies in different 293 sublineages, suggesting the viral genome structure is a factor for the sustainability of EBV's copy number. Moreover, the LMP1 transcription in high copy-containing cells showed abnormally high level. Furthermore, the main LMP1-driven pathway, transcription factor NF-κB, was highly activated in high-copy cells. Here we first manifest by experimental model that the copy number of EBV latent genome correlates with the viral pathogenesis, which depends on the activation level of LMP1 and NF-κB. Overall, both the presence and amount of EBV genome are crucial for the viral oncogenicity.

To obtain recombinant Epstein-Barr virus (EBV) particles with the neomycin resistance from BAC-EBV with hygrymicin (hyg) gene, homologous recombination was employed for altering the original hyg gene to neo resistance gene in the BAC-EBV genome. The kanamycin marker gene (kan) containing the recombinant protein FLP recognition sites (FRTs) was connected to the neo gene in the vector of pGEM-T using conventional cloning techniques. Both ends of the connective are the arms with 20bp of homology sequences responding to the ends next to the hyg gene. PCR and cloning techniques were further performed to extend homology arms to make them reach 89bp. The linear fragment kan-neo with 89bp of arms was then electroporated into the bacteria containing BAC-EBV genome. Under the mediation of the redαβγ system, the hyg gene in BAC-EBV was replaced by neo gene. A series of methods including antibiotic selection, PCR amplification, sequencing and restriction enzyme digestion were used for the identification of the mutated plasmids. The results showed that BAC-EBV/neo, was constructed as expected. Through the transfection of the BAC-EBV/neo plasmid, G418 selection, and GFP observation, we established 293-EBV/neo cell line. This research lays a foundation for the establishment of broader cell models in the study of EBV-associated pathogenic mechanisms.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for EBV episome's replication and mitotic segregation. During this process, EBNA1 may interact with a lot of host factors. Bimolecular complementation (BiMC) assay is a useful tool for a detection of protein-protein interaction in living cells. In the present study, a plasmid for expressing a fusion protein CYFP-EBNA1 was constructed. As expected, it could express CYFP-EBNA1 and localize in the cellular nuclear. The result also demonstrated that the plasmid is allowed to be used in the BiMC system. KEYWORD: Bimolecular complementation; Epstein-Barr virus nuclear antigen 1; Protein-protein interaction