Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2.

In Epstein-Barr virus (EBV) latent infection, the EBV-encoded RNAs EBER1 and EBER2 accumulate in the host cell nucleus to ~10(6) 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 ~10(6) 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.

Noncoding RNPs of viral origin.

Like their host cells, many viruses produce noncoding (nc)RNAs. These show diversity with respect to time of expression during viral infection, length and structure, protein-binding partners and relative abundance compared with their host-cell counterparts. Viruses, with their limited genomic capacity, presumably evolve or acquire ncRNAs only if they selectively enhance the viral life cycle or assist the virus in combating the host's response to infection. Despite much effort, identifying the functions of viral ncRNAs has been extremely challenging. Recent technical advances and enhanced understanding of host-cell ncRNAs promise accelerated insights into the RNA warfare mounted by this fascinating class of RNPs.

Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling.

The Epstein-Barr virus (EBV) noncoding RNAs, EBV-encoded RNA 1 (EBER1) and EBER2, are the most abundant viral transcripts in all types of latently infected human B cells, but their function remains unknown. We carried out heterokaryon assays using cells that endogenously produce EBERs to address their trafficking, as well as that of the La protein, because EBERs are quantitatively bound by La in vivo. Both in this assay and in oocyte microinjection assays, EBERs are confined to the nucleus, suggesting that their contribution to viral latency is purely nuclear. EBER1 does not bind exportin 5; therefore, it is unlikely to act by interfering with microRNA biogenesis. In contrast, La, which is a nuclear phosphoprotein, undergoes nucleocytoplasmic shuttling independent of the nuclear export protein Crm1. To ensure that small RNA shuttling can be detected in cells that are negative for EBER shuttling, we demonstrate the shuttling of U1 small nuclear RNA.

Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22.

EBER 1, a small noncoding viral RNA abundantly expressed in all cells transformed by Epstein-Barr virus (EBV), has been shown to associate with the human ribosomal protein L22. Here we present in vitro binding studies using purified RNAs and recombinant proteins. Electrophoretic mobility-shift assays (EMSAs) show that recombinant L22 (rL22) and maltose-binding protein (MBP)-tagged L22 protein bind EBER 1 in vitro, both forming three specific protein-dependent mobility shifts. Use of a mixture of rL22 and MBP-L22 indicates that these three shifts contain one, two, or three L22 proteins per EBER 1 molecule. EMSAs performed with EBER 1 deletion constructs and EBER 1 stem-loops inserted into a nonbinding RNA, HSUR 3, identify stem-loops I, III, and IV as L22 binding sites. The existence of multiple L22 binding sites on EBER 1 inside cells is demonstrated by in vivo UV cross-linking. Our results are discussed with respect to the function of EBER 1 in EBV-infected human B cells.