Abstract
Epstein-Barr virus (EBV) is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III). The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15), is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL) cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.
Highlights
Epstein-Barr virus (EBV) is a human gamma herpes virus carried by greater than 90% of the world’s population as a largely asymptomatic persistent latent infection in B-lymphocytes
To determine whether levels of RGC-32 fluctuated during the cell cycle, we examined RGC-32 protein expression in cell-cycle fractions obtained by centrifugal elutriation from Mutu III cells (Figure 1C)
We have provided the first demonstration that protein expression of the novel CDK1 activator, RGC-32, is upregulated in cells immortalized by a human tumour virus, further supporting a role for deregulated RGC-32 expression in tumour development
Summary
Epstein-Barr virus (EBV) is a human gamma herpes virus carried by greater than 90% of the world’s population as a largely asymptomatic persistent latent infection in B-lymphocytes. Immortalization of resting B cells by EBV in vitro leads to the generation of latently infected lymphoblastoid cell lines (LCLs) that express all EBV latent proteins: Epstein-Barr nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C, -LP and Latent membrane proteins (LMPs) 1, 2A and 2B, in addition to non-coding RNA species. This ‘full’ pattern of latent gene expression is termed latency III. Non-dividing EBV-positive cells lacking any latent gene expression have been detected in infected hosts (latency 0), demonstrating that infected cells can ‘shut-off’ latent gene expression when in a resting state [3]
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