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.
Highlights
Epstein-Barr virus (EBV) infection of primary B cells results in sustained cellular proliferation and immortalization of infected cells in vitro, through the expression of the viral latency-associated genes: EBV nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C and LP, latent membrane proteins (LMPs) 1, 2A and 2B, as well as a number of small noncoding viral RNAs and miRNAs, a program of viral gene expression known as Latency III
While tumors did not arise in mice injected with EBV-negative Akata Burkitt lymphoma (BL) cells, the average time to tumor development (2 cm in diameter) in mice injected with Latency I BL cells (Kem I) was 55 days, consistent with analysis of Latency I BLs in previous studies [53,54]
Recent studies that utilized a dominant-negative EBNA-1 to induce the loss of the EBV episome have expanded our understanding of the relative contributions of EBV to Wp-R and Latency I BL [78]
Summary
Epstein-Barr virus (EBV) infection of primary B cells results in sustained cellular proliferation and immortalization of infected cells in vitro, through the expression of the viral latency-associated genes: EBV nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C and LP, latent membrane proteins (LMPs) 1, 2A and 2B, as well as a number of small noncoding viral RNAs and miRNAs, a program of viral gene expression known as Latency III (reviewed in [1]). Selective expression of the viral genome-maintenance protein EBNA-1 in Latency I is required to sustain infection during periodic proliferation of these cells for their self-regeneration; Latency I is maintained by EBV-positive Burkitt lymphoma (BL) and BLderived cell lines. While Latency I is maintained in the majority of EBV-positive BL, Author Summary
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