title: EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival.
abstract: Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that can give rise to cancers of both B-cell and epithelial cell origin. In EBV-induced cancers of epithelial origin, including nasopharyngeal carcinomas (NPCs) and gastric carcinomas, the latent EBV genome expresses very high levels of a cluster of 22 viral pre-miRNAs, called the miR-BARTs, and these have previously been shown to confer a degree of resistance to pro-apoptotic drugs. Here, we present an analysis of the ability of individual miR-BART pre-miRNAs to confer an anti-apoptotic phenotype and report that five of the 22 miR-BARTs demonstrate this ability. We next used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to globally identify the mRNA targets bound by these miR-BARTs in latently infected epithelial cells. This led to the identification of ten mRNAs encoding pro-apoptotic mRNA targets, all of which could be confirmed as valid targets for the five anti-apoptotic miR-BARTs by indicator assays and by demonstrating that ectopic expression of physiological levels of the relevant miR-BART in the epithelial cell line AGS resulted in a significant repression of the target mRNA as well as the encoded protein product. Using RNA interference, we further demonstrated that knockdown of at least seven of these cellular miR-BART target transcripts phenocopies the anti-apoptotic activity seen upon expression of the relevant EBV miR-BART miRNA. Together, these observations validate previously published reports arguing that the miR-BARTs can exert an anti-apoptotic effect in EBV-infected epithelial cells and provide a mechanistic explanation for this activity. Moreover, these results identify and validate a substantial number of novel mRNA targets for the anti-apoptotic miR-BARTs.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America; Center for Virology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Dong
authors.initials: D
authors.lastname: Kang
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America; Center for Virology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Skalsky
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America; Center for Virology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
mesh_terms: D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D020031:Epstein-Barr Virus Infections / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D057131:Immune Evasion / Q000235:genetics*; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000235:genetics*; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012367:RNA, Viral / Q000235:genetics*; D017423:Sequence Analysis, RNA; D014162:Transfection; D017735:Virus Latency / Q000235:genetics*
chemical_list: D035683:MicroRNAs; D012367:RNA, Viral