Abstract
In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~106 copies. While the expression of EBERs in cell lines is associated with transformation, a mechanistic explanation of their roles in EBV latency remains elusive. To identify EBER-specific gene expression features, we compared the proteome and mRNA transcriptome from BJAB cells (an EBV-negative B lymphoma cell line) stably transfected with an empty plasmid or with one carrying both EBER genes. We identified ~1800 proteins with at least 2 SILAC pair measurements, of which only 8 and 12 were up- and downregulated ≥ 2-fold, respectively. One upregulated protein was PIK3AP1, a B-cell specific protein adapter known to activate the PI3K-AKT signaling pathway, which regulates alternative splicing and translation in addition to its pro-survival effects. In the mRNA-seq data, the mRNA levels for some of the proteins changing in the SILAC data did not change. We instead observed isoform switch events. We validated the most relevant findings with biochemical assays. These corroborated the upregulation of PIK3AP1 and AKT activation in BJAB cells expressing high levels of both EBERs and EBNA1 (a surrogate of Burkitt’s lymphoma EBV latency I) relative to those expressing only EBNA1. The mRNA-seq data in these cells showed multiple upregulated oncogenes whose mRNAs are enriched for 3´-UTR AU-rich elements (AREs), such as ccl3, ccr7, il10, vegfa and zeb1. The CCL3, CCR7, IL10 and VEGFA proteins promote cell proliferation and are associated with EBV-mediated lymphomas. In EBV latency, ZEB1 represses the transcription of ZEBRA, an EBV lytic phase activation factor. We previously found that EBER1 interacts with AUF1 in vivo and proposed stabilization of ARE-containing mRNAs. Thus, the ~106 copies of EBER1 may promote not only cell proliferation due to an increase in the levels of ARE-containing genes like ccl3, ccr7, il10, and vegfa, but also the maintenance of latency, through higher levels of zeb1.
Highlights
Epstein-Barr virus (EBV) is a human γ-herpesvirus that infects primarily B cells, establishing a life-long latent infection that is innocuous in most healthy individuals; but in those with immunodeficiency, infection can cause cancer [1, 2]
We found that the activation of BJAB cells by either high-level EBER expression (BJAB-EBNA1-EBER1/2), EBNA1 alone (BJAB-EBNA1) or EBV infection (BJAB-B1), increases the proliferation rate of these cells, when compared to the parental BJAB control (Fig 1E)
We could not validate the upregulation of these proteins by western blot (WB) due to poor antibody quality, we found that the total mRNA levels of both genes were upregulated in BJAB-EBER1/2 cells to a similar extent compared to their SILAC ratio (Fig 4D and Table 1)
Summary
EBV is a human γ-herpesvirus that infects primarily B cells, establishing a life-long latent infection that is innocuous in most healthy individuals; but in those with immunodeficiency, infection can cause cancer [1, 2]. In addition to the latent viral protein EBNA1, the EBV noncoding (nc) RNAs, EBER1 and EBER2, are expressed at high levels in all four latency stages [1, 4]. The EBERs are transcribed by RNA Polymerase III, lack a poly-A tail and were first described in EBV-infected B cells as the RNA components of ribonucleoprotein (RNP) complexes that include the cellular autoantigen La [5, 6]. Their nucleotide sequences are ~50% identical and their experimentally-defined secondary structures contain multiple stems and loops [7]. We have shown using stable isotope labeling of amino acids in cell culture (SILAC) proteomics that EBER1 interacts with various hnRNPs, including the AU-rich element (ARE)-binding protein AUF1 (hnRNP D) [10]
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