Epstein-Barr Virus Nuclear Antigen 3A Promotes Cellular Proliferation by Repression of the Cyclin-Dependent Kinase Inhibitor p21WAF1/CIP1

Abstract

Latent infection by Epstein-Barr virus (EBV) is highly associated with the endemic form of Burkitt lymphoma (eBL), which typically limits expression of EBV proteins to EBNA-1 (Latency I). Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R) - that includes expression of the EBNA-3 proteins (3A, 3B and 3C), in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb) to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs), consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14ARF and p16INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14ARF and p16INK4a. By contrast, p16INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to the proliferation of Wp-R BL cells and LCLs.

Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2

“…We, and others, had previously shown that EBNA3A and EBNA3C can block transcription of two members of the INK4 CDKI family, p16 INK4a and p15 INK4b [ 12 , 30 , 32 , 78 ]. Furthermore it has been reported that these same two EBV proteins might also repress expression of a third member of the CIP/KIP family, p21 CIP1 [ 85 , 86 ]. Since intrinsic cell cycle inhibitors are emerging as important targets of EBNA3A and EBNA3C, we focused on the consequences of miR-221/miR-222 induction in LCLs.…”

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

“…In LCLs and Burkitt lymphoma tumor cells, the EBNA3 proteins have been shown to regulate distinct but extensively overlapping sets of cell genes (19)(20)(21)(22)(23)(24)(25)(26). EBNA3 proteins have been implicated in the pathogenesis of Burkitt lymphoma and in attenuating an antiproliferative DNA damage response during EBV transformation of primary B lymphocytes (19,(27)(28)(29). Moreover, EBNA3A and EBNA3C repression of the CDKN2A-encoded tumor suppressors p16 and p14 is essential for LCL growth, requires interaction with RBPJ, and is associated with increased H3K27me3 modification at the CDKN2A promoter (22,23,(30)(31)(32).…”

Epstein–Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes

“…GSE59181). (29,30). EBNA3A initiates and maintains polycomb repressive signatures at the CXCL9 and CXCL10 loci (31), and EBNA3A expression in BL cells represses proapoptotic Bim expression (32).…”