title: RNase L targets distinct sites in influenza A virus RNAs.
abstract: Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled.
  
  
  OBJECTIVE
  RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.
authors.affiliation: Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Daphne A
authors.initials: DA
authors.lastname: Cooper
authors.affiliation: Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Shuvojit
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Arindam
authors.initials: A
authors.lastname: Chakrabarti
authors.affiliation: Department of Microbiology, Department of Medicine, Division of Infectious Diseases, Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado, USA Program in Molecular Biology, University of Colorado School of Medicine, Aurora, Colorado, USA.
authors.forename: Jay R
authors.initials: JR
authors.lastname: Hesselberth
authors.affiliation: Department of Cancer Biology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Silverman
authors.affiliation: Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, Colorado, USA Program in Molecular Biology, University of Colorado School of Medicine, Aurora, Colorado, USA david.barton@ucdenver.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Barton
mesh_terms: D002460:Cell Line; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000821:virology; D055785:Gene Knockdown Techniques; D059014:High-Throughput Nucleotide Sequencing; D054884:Host-Pathogen Interactions; D006801:Humans; D006868:Hydrolysis; D009980:Influenza A virus / Q000276:immunology / Q000502:physiology*; D020871:RNA Stability; D012367:RNA, Viral / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease