title: Proteomics and Transcriptomics of BJAB Cells Expressing the Epstein-Barr Virus Noncoding RNAs EBER1 and EBER2.
abstract: 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Genaro
authors.initials: G
authors.lastname: Pimienta
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Victor
authors.initials: V
authors.lastname: Fok
authors.affiliation: Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Maria
authors.initials: M
authors.lastname: Haslip
authors.affiliation: Department of Genetics, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Maria
authors.initials: M
authors.lastname: Nagy
authors.affiliation: Section of Pulmonary, Critical Care, and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Seyedtaghi
authors.initials: S
authors.lastname: Takyar
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, United States of America.
authors.forename: Joan A
authors.initials: JA
authors.lastname: Steitz
mesh_terms: D045744:Cell Line, Tumor; D020031:Epstein-Barr Virus Infections / Q000821:virology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016393:Lymphoma, B-Cell / Q000821:virology; D009857:Oncogenes; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D017735:Virus Latency / Q000235:genetics
chemical_list: D019309:Epstein-Barr Virus Nuclear Antigens; C099537:Epstein-Barr virus encoded RNA 1; C106732:Epstein-Barr virus encoded RNA 2; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D022661:RNA, Untranslated; D012367:RNA, Viral; D014764:Viral Proteins; C417029:EBV-encoded nuclear antigen 1