Epstein-Barr Virus (EBV) Nuclear Protein 2-Induced Disruption of EBV Latency in the Burkitt’s Lymphoma Cell Line Akata: Analysis by Tetracycline-Regulated Expression

Abstract

The Burkitt’s lymphoma (BL) cell line Akata retains the latency I program of Epstein-Barr virus (EBV) gene expression and cross-linking of its surface immunoglobulin G (IgG) by antibodies results in activation of viral replication. When EBV nuclear antigen 2 (EBNA2) was artificially expressed by a constitutive expression vector, the Cp EBNA promoter remained inactive and accordingly the latency III program was not induced. In contrast, expression of LMP2A and activity of the Fp lytic promoter were activated. Consistent with this Fp activity, the rate of spontaneous activation of the EBV replicative cycle was increased significantly, suggesting the possibility that EBNA2 can induce EBV replication. The efficiency of anti-IgG-induced activation of the viral replication was reduced in Akata cells expressing EBNA2. To obtain more direct evidence for EBNA2-induced activation of the EBV replicative cycle, this protein was next expressed by a tetracycline-regulated expression system. EBNA2 was undetectable with low doses (<0.5 μg/ml) of tetracycline, while its expression was rapidly induced after removal of the antibiotic. This induced expression of EBNA2 was immediately followed by expression of EBV replicative cycle proteins in up to 50% of the cells, as shown by indirect immunofluorescence and immunoblot analysis. These results suggest an unexpected potential of EBNA2 to disrupt EBV latency and to activate viral replication.

Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

“…An increase in LMP2 expression was also observed in Akata cells by Southern reverse transcription PCR [ 25 ], but our analysis of the kinetics of latent EBV genes transcription indicates that their transcription is increased concomitantly with early lytic EBV genes. EBNA2 can induce lytic infection in Akata cells under some circumstances [ 26 ], but LMP1 [ 27 ] and LMP2 [ 28 ] exert a negative effect on induction of lytic infection. From the microarray data, one might hypothesize a novel mechanism in which transcription of latent genes regulates activation of lytic infection.…”

“…To characterize quantitative EBV gene transcription, we chose a panel of EBV-harboring BL cell lines with different characteristics. The panel included three BL cell lines with EBV type I virus: Namalwa [ 29 ], Raji [ 30 ] (50 copies of EBV per cell [ 31 ] with a deletion in EBNA3-C [ 32 ], and Akata [ 26 ] (20 copies of EBV per cell). It also contained two BL cell lines with EBV type II virus: Jijoye and its daughter cell line P3HR1, which has a deletion in EBNA2.…”