Epstein–Barr virus facilitates the malignant potential of immortalized epithelial cells: from latent genome to viral production and maintenance

Abstract

Epstein-Barr virus (EBV) is closely associated with several malignancies, including nasopharyngeal carcinoma. To investigate the EBV activity in tumor development, we tried to establish a malignant model of EBV-infected cells in nude mice. On the basis of the Maxi-EBV system, a human embryonic kidney epithelial cell line (293) with a low malignant potential was used for a stable EBV genome infection. The derived cell line, termed 293-EBV, exhibited obvious morphological transformation and significantly increased growth ability, with the cell cycle redistributed. The clonability and tumorigenicity were also substantially accelerated. In 293-EBV cells, the expression level of the transcription factor NF-kB and JNK2 were upregulated. The result suggested that latent membrane protein 1 (LMP1) was an important viral protein responsible for the enhanced malignant potential. Matured and budding virus particles were observed in tumor tissues, confirming the spontaneous reactivation of EBV from latent genome to lytic cycle at the site of tumor development. Primary culture of tumor tissues showed two patterns about the EBV maintenance or not in newly grown cells, and this was dependent on the thickness of the planted tissues. Moreover, the tumor cells lost EBV genome easily when subcultured at low density. Our findings revealed the cell-to-cell contact mechanism, which was required for the EBV maintenance in the tumor cells during the expansion of EBV-infected cells. This mechanism might give an explanation to the phenomenon that EBV genome in epithelial tumor cells becomes easily lost during subculture in vitro. Our results provided further evidence of a function for EBV in the etiology of tumor development.

SPLUNC1 reduces the inflammatory response of nasopharyngeal carcinoma cells infected with the EB virus by inhibiting the TLR9/NF-κB pathway

“…HEK-293 cells stably transfected with the p2089 plasmid (293-EBV) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (Therm, UK) (12). An immortalized normal nasopharyngeal epithelial cell line NP69 was cultured in keratinocyte serum-free medium (gifted by the Chinese University of Hong Kong) (13).…”

“…The viral genes BZLF1 and BALF4 in the pcDNA3.1(+) vector were transiently transfected into the 293-EBV cells (12,16), and the GFP-EBV was released from the transfected cells into the supernatant due to expression of BZLF1 and BALF4 which was induced by lytic replication of the 293-EBV cells (17). After 72 h, the supernatant was collected and filtered through a 0.45-mm pore-size filter.…”

“…Through the cell-to-cell contact method in our laboratory as previously described (12,18), three human NPC cell lines (highly differentiated CNE1, poorly differentiated HNE2 and undifferentiated 5-8F cells) were infected with recombinant GFP-EBV produced from the 293-EBV cells. Briefly, Raji cells were exposed to EBV produced from 293-EBV cells that were induced with the expression plasmids BZLF1 and BALF4 for 3 h at 4˚C.…”

Establishment of Bimolecular Complementation System for the Interaction Detection between Epstein-barr Virus Nuclear Antigen 1 and Host Proteins

“…Western blotting were performed as described previously [8]. Protein samples were subject to electrophoresis in SDS-PAGE gels and analyzed ABSTRACT: Epstein-Barr virus nuclear antigen 1 (EBNA1) is essential for EBV episome's replication and mitotic segregation.…”

Nasopharyngeal carcinoma: Advances in genomics and molecular genetics

“…This method provided the basis for whole-genome level investigations into the mechanism by which EBV contributes to NPC development [104]. We found that infection with the whole genome of EBV significantly promoted the growth and malignancy of epithelial cells [105] and activated NF-κB and MAPK signaling [106].…”