title: Systematic microRNA analysis identifies ATP6V0C as an essential host factor for human cytomegalovirus replication.
abstract: Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions.
authors.affiliation: Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Jon
authors.initials: J
authors.lastname: Pavelin
authors.affiliation: Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Natalie
authors.initials: N
authors.lastname: Reynolds
authors.affiliation: Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Stephen
authors.initials: S
authors.lastname: Chiweshe
authors.affiliation: OCTRI, Health Sciences University, Portland, Oregon, United States of America.
authors.forename: Guanming
authors.initials: G
authors.lastname: Wu
authors.affiliation: Vaccine and Gene Therapy Institute, Health Sciences University, Portland, Oregon, United States of America.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Tiribassi
authors.affiliation: Division of Infection and Immunity, The Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom.
authors.forename: Finn
authors.initials: F
authors.lastname: Grey
mesh_terms: D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000472:pathogenicity / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000235:genetics; D020869:Gene Expression Profiling; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D046228:Microarray Analysis; D030781:Organisms, Genetically Modified; D034741:RNA, Small Interfering / Q000494:pharmacology; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics*
chemical_list: C541389:ATP6V0C protein, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; D014764:Viral Proteins; D025262:Vacuolar Proton-Translocating ATPases