The Impact of Deleting Stem-Loop 1 of Epstein-Barr Virus-Encoded RNA 1 on Cell Proliferation

Epstein-Barr virus-encoded RNAs (EBERs) are two small, noncoding, structurally conserved transcripts, constitutively expressed at >10(6) copies per EBV-infected cell. They have been shown to drive cell growth. However, the mechanism(s) involved in EBER-induced proliferation is not clear. In this study, we investigated the molecular mechanisms and structural impact of EBER1. Sequences of EBER1 stem-loops (SL) 1, 3, and 4 were deleted, creating three mutants: increment SL1, increment SL3, and increment SL4. These mutants were cloned into pHebo plasmids and expressed in Jurkat cell lines. Cells transfected with wildtype EBER1 and pHebo were used as controls. Cell proliferation was monitored by microscopy and flow cytometry. Microarray, qPCR, and Western blotting were used to investigate the cell cycle markers. We found significantly higher cell proliferation in wildtype EBER1 cells compared to pHebo, increment SL1, and increment SL3, but not increment SL4 mutants. There was also significant upregulation of S-phase and G2/M phase markers in wildtype EBER1 and increment SL4 mutant. Furthermore, CDT1, a factor for DNA replication, was upregulated in wildtype EBER1 and increment SL4 mutant. However, in increment SL1 mutant, CDT1 was significantly downregulated and translocated to the cytoplasm. These data indicate that the structure of EBER1 is important in cell proliferation.