title: Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein.
abstract: Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ-induced responses, for example, expression of IFN-stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress-induced-phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca(2+) levels while inhibiting nuclear translocalization of STAT1 and NFAT-1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca(2+) /calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5-mediated inhibition of IFNβ-induced responses, for example, IFN-sensitive response element driven luciferase, STAT1-dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5-expressing cells in response to IFNβ. Results prove Ca(2+) , calreticulin, and calcineurin involvement in STAT1-mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.
authors.affiliation: Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming University, Taipei, Taiwan.
authors.forename: Tsuey-Ching
authors.initials: TC
authors.lastname: Yang
authors.forename: Shih-Wein
authors.initials: SW
authors.lastname: Li
authors.forename: Chien-Chen
authors.initials: CC
authors.lastname: Lai
authors.forename: Kai-Zen
authors.initials: KZ
authors.lastname: Lu
authors.forename: Man-Tzu
authors.initials: MT
authors.lastname: Chiu
authors.forename: Tsung-Han
authors.initials: TH
authors.lastname: Hsieh
authors.forename: Lei
authors.initials: L
authors.lastname: Wan
authors.forename: Cheng-Wen
authors.initials: CW
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D016572:Cyclosporine / Q000494:pharmacology; D004664:Encephalitis Virus, Japanese / Q000502:physiology*; D005786:Gene Expression Regulation / Q000276:immunology; D054884:Host-Pathogen Interactions; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D016899:Interferon-beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D050794:STAT1 Transcription Factor / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; C096041:NS5 protein, flavivirus; D020543:Proteome; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D017361:Viral Nonstructural Proteins; D016899:Interferon-beta; D016572:Cyclosporine; D019703:Calcineurin
keywords: Calreticulin; Interferon; Japanese encephalitis virus; Microbiology; Non-structural protein 5; STAT1