Abstract
Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the BamHI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, BamHI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates Qp-EBNA1 expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, while electrophoretic mobility shift assays (EMSAs) demonstrated that p50 can also bind to Qp. Inhibition of NF-κB signaling by the IκB kinase inhibitor PS-1145 resulted in the downregulation of Qp-EBNA1 expression in C666-1 NPC cells. Since EBNA1 has been reported to block p65 activation by inhibiting IKKα/β through an unknown mechanism, we suggest that, in NPC, NF-κB signaling and EBNA1 may form a regulatory loop which supports EBV latent gene expression, while also limiting NF-κB activity. These findings emphasize the role of NF-κB signaling in the regulation of EBV latency in EBV-associated tumors.
Highlights
Epstein–Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is able to establish lifelong latent infection in vivo
Both mutated Q promoter (Qp) reporters showed significantly reduced responsiveness to activation by p65 compared to the wild type Qp reporter (Figure 1D), further confirming the involvement of nuclear factor-κB (NF-κB) in Qp activation. Both NF-κB half sites seemed to be involved in p65-mediated activation of Qp. These results demonstrate that the EBV Qp promoter contains an NF-κB binding site which is positively regulated by NF-κB signaling
Immunoblot analysis of cellular fractions of C666-1 cells revealed that p50 was present at similar levels in the cytoplasm and nucleus, while only a relatively small fraction of p65 could be detected in the nucleus (Figure 4B). Both Bcl3 and STAT3 were highly expressed in C666-1 cells, but were completely restricted to the cytoplasm, while IRF2 was detectable in the nucleus (Figure 4B). These results demonstrate that both p65 and p50 NF-κB subunits are present in the nucleus of EBV-harboring nasopharyngeal carcinoma (NPC) cell line C666-1
Summary
Epstein–Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is able to establish lifelong latent infection in vivo. During EBV latency in vivo, viral gene expression is tightly regulated, with very few essential viral antigens being expressed, while the viral genome is stably maintained as cells divide [2]. EBVs in different associated tumors exhibit different latent protein expression patterns, Epstein–Barr nuclear antigen 1 (EBNA1) expression is always present. Cancers 2018, 10, 119 and maintenance of the episomal EBV genome through binding to the plasmid origin of viral replication (oriP) [3]. EBNA1 binds to other viral gene promoters and functions as a transcriptional transactivator to regulate the expression of latent membrane protein 1 (LMP1) and other EBNAs [4]
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