title: Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription.
abstract: Coronaviruses contain a positive-sense single-stranded genomic (g) RNA, which encodes nonstructural proteins. Several subgenomic mRNAs (sgmRNAs) encoding structural proteins are generated by template switching from the body transcription regulatory sequence (TRS) to the leader TRS. The process preferentially generates shorter sgmRNA. Appropriate readthrough of body TRSs is required to produce longer sgmRNAs and full-length gRNA. We find that phosphorylation of the viral nucleocapsid (N) by host glycogen synthase kinase-3 (GSK-3) is required for template switching. GSK-3 inhibition selectively reduces the generation of gRNA and longer sgmRNAs, but not shorter sgmRNAs. N phosphorylation allows recruitment of the RNA helicase DDX1 to the phosphorylated-N-containing complex, which facilitates template readthrough and enables longer sgmRNA synthesis. DDX1 knockdown or loss of helicase activity markedly reduces the levels of longer sgmRNAs. Thus, coronaviruses employ a unique strategy for the transition from discontinuous to continuous transcription to ensure balanced sgmRNAs and full-length gRNA synthesis.
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, No. 1, Jen-Ai Road, Section 1, Taipei 10051, Taiwan.
authors.forename: Chia-Hsin
authors.initials: CH
authors.lastname: Wu
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, No. 1, Jen-Ai Road, Section 1, Taipei 10051, Taiwan; Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, No. 1, Jen-Ai Road, Section 1, Taipei 10051, Taiwan; National Taiwan University Research Center for Medical Excellence, No. 2, Syu-Jhou Road, Taipei 10055, Taiwan.
authors.forename: Pei-Jer
authors.initials: PJ
authors.lastname: Chen
authors.affiliation: Department of Microbiology, National Taiwan University College of Medicine, No. 1, Jen-Ai Road, Section 1, Taipei 10051, Taiwan; National Taiwan University Research Center for Medical Excellence, No. 2, Syu-Jhou Road, Taipei 10055, Taiwan; Department of Laboratory Medicine, National Taiwan University Hospital, No. 1, Changde Street, Taipei 10048, Taiwan. Electronic address: shyeh@ntu.edu.tw.
authors.forename: Shiou-Hwei
authors.initials: SH
authors.lastname: Yeh
mesh_terms: D000818:Animals; D002460:Cell Line; D017934:Coronavirus / Q000502:physiology*; D000086462:Coronavirus Nucleocapsid Proteins; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D051379:Mice; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D020365:RNA Helicases; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D014158:Transcription, Genetic
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D038362:Glycogen Synthase Kinase 3; C541494:DDX1 protein, mouse; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases