title: The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing.
abstract: The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing.
  
  
  METHODS
  We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model.
  
  
  RESULTS
  We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect.
  
  
  CONCLUSIONS
  The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address: wlin1@mgh.harvard.edu.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Lin
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; Department of Infectious Disease, Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui 230001, China.
authors.forename: Chuanlong
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Hong
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA; Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Jilg
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Dahlene N
authors.initials: DN
authors.lastname: Fusco
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Esperance A
authors.initials: EA
authors.lastname: Schaefer
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Brisac
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Lee F
authors.initials: LF
authors.lastname: Peng
authors.affiliation: Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Qikai
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Liver Center and Gastrointestinal Division, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. Electronic address: rtchung@mgh.harvard.edu.
authors.forename: Raymond T
authors.initials: RT
authors.lastname: Chung
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics*; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D055785:Gene Knockdown Techniques; D016174:Hepacivirus / Q000502:physiology*; D006526:Hepatitis C / Q000235:genetics / Q000821:virology; D006801:Humans; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha / Q000378:metabolism / Q000494:pharmacology; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects; D017381:Spliceosomes / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology
chemical_list: D000951:Antigens, Neoplasm; D000998:Antiviral Agents; D050834:Interferon-Stimulated Gene Factor 3, gamma Subunit; D016898:Interferon-alpha; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017411:Ribonucleoproteins, Small Nuclear; C431323:SART1 protein, human
keywords: Hepatitis C virus (HCV); Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway; RNA splicing; RNA-Sequencing (RNA-Seq); Squamous cell carcinoma antigen recognized by T cells (SART1)