Expression of LMP and EBNA genes in Epstein-Barr virus-associated lymphomas in Hu-PBL/SCID mice.

Transplantation of peripheral blood lymphocytes (PBLs) from healthy humans with latent Epstein-Barr virus (EBV) infection into severe combined immunodeficiency (SCID) mice results in development of EBV-associated human B-cell lymphoma. However, the expression of EBV genes in relation to lymphoma development has not been reported. We investigated latent membrane protein (LMP) and EBV nuclear antigen (EBNA) gene expression in PBLs from EBV-positive blood donors and induced-lymphoma cells from SCID mice to elucidate the functions and effects of the EBV genome in the occurrence and development of lymphoma. PBLs were isolated from 9 healthy blood donors and transplanted into SCID mice. Gene expression levels of LMP-1, LMP-2A, and LMP-2B and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP were monitored by real-time quantitative-polymerase chain reaction (qRT-PCR) in cells from nine EBV-induced lymphomas and in matched lymphocytes from healthy subjects. LMP-1, EBNA-1 and EBNA-2 protein levels were detected by western blotting. As a result, LMP-1, LMP-2A and LMP-2B mRNA levels were upregulated 256-, 38- and 331-fold, respectively, in the EBV-induced lymphoma cells compared with the controls, while EBNA-1 and EBNA-3A mRNA levels were upregulated 1157- and 1154-fold, respectively. EBNA-2, EBNA-3B, EBNA-3C and EBNA-LP mRNAs were detected in lymphoma cells, but not in lymphocytes from EBV-positive blood donors. LMP-1 and EBNA-2 proteins were not expressed in lymphocytes from EBV-positive blood donors, according to western blotting. Weak EBNA-1 expression was observed in lymphocytes from blood donors with latent EBV infection, while LMP-1, EBNA-1 and EBNA-2 protein levels were significantly upregulated in EBV-induced lymphoma cells, consistent with mRNA expression levels detected by qRT-PCR. In conclusion, LMP-1, LMP-2A, LMP-2B, EBNA-1 and EBNA-3A were upregulated in EBV-induced lymphoma cells, while EBNA-2, EBNA-3B, EBNA-3C and EBNA-LP were absent in lymphocytes from humans with latent EBV infection, but were positively expressed in EBV-induced lymphoma cells.

Expression of latent membrane proteins in Epstein­Barr virus-transformed lymphocytes in vitro.

Infection with Epstein-Barr virus (EBV) induces activation and proliferation of B lymphocytes. Detection of latent membrane protein (LMP)-1 is used to identify the proliferative ability of B cells. However, changes in the expression levels of the three LMPs during EBV-induced B lymphocyte transformation, have not yet been reported. In the present study, the expression levels of LMP-1, LMP-2A and LMP-2B were compared between EBV-transformed B lymphocytes and paired normal lymphocytes. Seven lymphoblast cell lines were established by EBV infection of normal human lymphocytes in vitro. The expression levels of LMP genes and LMP-1 protein were determined using quantitative (q)PCR and western blotting in lymphoblasts and normal lymphocytes, respectively. The expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts compared with the normal lymphocytes. The LMP-1 protein level was also significantly increased in EBV-transformed B lymphocytes. Expression of LMP1, LMP-2A and LMP-2B genes was significantly upregulated in EBV-induced lymphoblasts, suggesting LMP genes are important in the transformation of human lymphocytes.