Epstein-Barr virus nuclear antigens 3C and 3A maintain lymphoblastoid cell growth by repressing p16INK4A and p14ARF expression

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and EBNA3A are each essential for EBV conversion of primary human B lymphocytes into continuously proliferating lymphoblast cell lines (LCLs) and for maintaining LCL growth. We now find that EBNA3C and EBNA3A's essential roles are to repress p16INK4A and p14ARF. In the absence of EBNA3C or EBNA3A, p16INK4A and p14ARF expression increased and cell growth ceased. EBNA3C inactivation did not alter p16INK4A promoter CpG methylation, but reduced already low H3K27me3, relative to resting B cells, and increased H3K4me3 and H3-acetylation, linking EBNA3C inactivation to histone modifications associated with increased transcription. Importantly, knockdown of p16INK4A or p14ARF partially rescued LCLs from EBNA3C or EBNA3A inactivation-induced growth arrest and knockdown of both rescued LCL growth, confirming central roles for p16INK4A and p14ARF in LCL growth arrest following EBNA3C or EBNA3A inactivation. Moreover, blockade of p16INK4A and p14ARF effects on pRb and p53 by human papilloma virus type 16 E7 and E6 expression, sustained LCL growth after EBNA3C or EBNA3A inactivation. These data indicate that EBNA3C and EBNA3A joint repression of CDKN2A p16INK4A and p14ARF is essential for LCL growth.