title: Identification of immediate early gene X-1 as a cellular target gene of hcmv-mir-UL148D.
abstract: Human cytomegalovirus (HCMV) is a herpesvirus that causes congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behavior. The identification of functional target genes of miRNAs is an important step in the study of HCMV pathogenesis. HCMV encodes at least 14 miRNAs, including hcmv-mir-UL148D, which resides in the HCMV UL/b' region. hcmv-mir-UL148D is the only miRNA encoded by the HCMV UL/b' region; however, its targets and functional effects have not yet been eludidated. In this study, hybrid-PCR screening was used to identify target genes and dual luciferase reporter assay was used to evaluate the binding effect of hcmv-miR-UL148D to the 3' untranslated region (3'UTR) of IEX-1. In addition, western blot analysis was used to detect the expression kinetics of IEX-1 protein and apoptosis assay was used to identify the effects of hcmv-miR-UL148D on cell apoptosis. The hybrid-PCR results showed that only one binding site in the 3'UTR of the cellular gene, human immediate early gene X-1 (IEX-1), was completely complementary to an 11 nucleotide (nt) sequence in the 5' terminus of hcmv-mir-UL148D, including the entire seed region. The binding site was demonstrated to be functional by dual luciferase reporter assay with a 47% repression of the relative luciferase activity. In an in vitro system of human embryonic kidney 293 (HEK293) cells, the ectopically expressed hcmv-mir-UL148D exhibited a downregulatory effect on IEX-1 expression, and decreased the cell apoptosis induced by transfected IEX-1. Our data demonstrate that hcmv-mir-UL148D targets the cellular gene, IEX-1, downregulating its expression and thus results in anti-apoptotic effects.
authors: Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, PR China.
authors: Yue-Ping
authors: YP
authors: Wang
authors: Ying
authors: Y
authors: Qi
authors: Yu-Jing
authors: YJ
authors: Huang
authors: Man-Long
authors: ML
authors: Qi
authors: Yan-Ping
authors: YP
authors: Ma
authors: Rong
authors: R
authors: He
authors: Yao-Hua
authors: YH
authors: Ji
authors: Zheng-Rong
authors: ZR
authors: Sun
authors: Qiang
authors: Q
authors: Ruan
mesh_terms: D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism; D003586:Cytomegalovirus Infections / Q000378:metabolism / Q000821:virology; D015536:Down-Regulation; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C099815:IER3 protein, human; D008565:Membrane Proteins; D035683:MicroRNAs; D012367:RNA, Viral