title: Host MicroRNA miR-197 Plays a Negative Regulatory Role in the Enterovirus 71 Infectious Cycle by Targeting the RAN Protein.
abstract: Enterovirus 71 (EV71), a member of Picornaviridae, is associated with severe central nervous system complications. In this study, we identified a cellular microRNA (miRNA), miR-197, whose expression was downregulated by viral infection in a time-dependent manner. In miR-197 mimic-transfected cells, EV71 replication was inhibited, whereas the internal ribosome entry site (IRES) activity was decreased in EV71 strains with or without predicted miR-197 target sites, indicating that miR-197 targets host proteins to modulate viral replication. We thus used a quantitative proteomics approach, aided by the TargetScan algorithm, to identify putative target genes of miR-197. Among them, RAN was selected and validated as a genuine target in a 3' untranslated region (UTR) reporter assay. Reduced production of RAN by RNA interference markedly reduced the synthesis of EV71-encoded viral proteins and virus titers. Furthermore, reintroduction of nondegradable RAN into these knockdown cells rescued viral protein synthesis. miR-197 levels were modulated by EV71 to maintain RAN mRNA translatability at late times postinfection since we demonstrated that cap-independent translation exerted by its intrinsic IRES activity was occurring at times when translation attenuation was induced by EV71. EV71-induced downregulation of miR-197 expression increased the expression of RAN, which supported the nuclear transport of the essential viral proteins 3D/3CD and host protein hnRNP K for viral replication. Our data suggest that downregulation of cellular miRNAs may constitute a newly identified mechanism that sustains the expression of host proteins to facilitate viral replication.
  
  
  OBJECTIVE
  Enterovirus 71 (EV71) is a picornavirus with a positive-sense single-stranded RNA that globally inhibits the cellular translational system, mainly by cleaving cellular eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein (PABP), which inhibits the association of the ribosome with the host capped mRNA. We used a microRNA (miRNA) microarray chip to identify the host miRNA 197 (miR-197) that was downregulated by EV71. We also used quantitative mass spectrometry and a target site prediction tool to identify the miR-197 target genes. During viral infection, the expression of the target protein RAN was upregulated considerably, and there was a parallel downregulation of miR-197. The nuclear transport of viral 3D/3CD protein and of the host proteins involved in viral replication proceeded in an RAN-dependent manner. We have identified a new mechanism in picornavirus through which EV71-induced cellular miRNA downregulation can regulate host protein levels to facilitate viral replication.
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Wen-Fang
authors.initials: WF
authors.lastname: Tang
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Ru-Ting
authors.initials: RT
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Kun-Yi
authors.initials: KY
authors.lastname: Chien
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Jo-Yun
authors.initials: JY
authors.lastname: Huang
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Kean-Seng
authors.initials: KS
authors.lastname: Lau
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Jia-Rong
authors.initials: JR
authors.lastname: Jheng
authors.affiliation: Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan.
authors.forename: Cheng-Hsun
authors.initials: CH
authors.lastname: Chiu
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan.
authors.forename: Tzong-Yuan
authors.initials: TY
authors.lastname: Wu
authors.affiliation: Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan.
authors.forename: Chung-Yung
authors.initials: CY
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology and Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Taoyuan, Taiwan Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan jimtong@mail.cgu.edu.tw.
authors.forename: Jim-Tong
authors.initials: JT
authors.lastname: Horng
mesh_terms: D029821:Enterovirus A, Human / Q000276:immunology* / Q000502:physiology*; D005786:Gene Expression Regulation; D054884:Host-Pathogen Interactions; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis*; D014779:Virus Replication; D020931:ran GTP-Binding Protein / Q000378:metabolism*
chemical_list: C546085:MIRN197 microRNA, human; D035683:MicroRNAs; C120587:RAN protein, human; D014764:Viral Proteins; D020931:ran GTP-Binding Protein