title: Hantavirus GnT elements mediate TRAF3 binding and inhibit RIG-I/TBK1-directed beta interferon transcription by blocking IRF3 phosphorylation.
abstract: Hantaviruses successfully replicate in primary human endothelial cells by restricting the early induction of beta interferon (IFN-β) and interferon-stimulated genes (ISGs). Gn proteins from NY-1V, ANDV, and TULV, but not PHV, harbor elements in their 142-residue cytoplasmic tails (GnTs) that inhibit RIG-I/MAVS/TBK1-TRAF3-directed IFN-β induction. Here, we define GnT interactions and residues required to inhibit TRAF3-TBK1-directed IFN-β induction and IRF3 phosphorylation. We observed that GnTs bind TRAF3 via residues within the TRAF-N domain (residues 392 to 415) and that binding is independent of the MAVS-interactive TRAF-C domain (residues 415 to 568). We determined that GnT binding to TRAF3 is mediated by C-terminal degrons within NY-1V or ANDV GnTs and that mutations that add degrons to TULV or PHV GnTs confer TRAF3 binding. Further analysis of GnT domains revealed that TRAF3 binding is a discrete GnT function, independent of IFN regulation, and that residues 15 to 42 from the NY-1V GnT C terminus are required for inhibiting TBK1-directed IFN-β transcription. Mutagenesis of the NY-1V GnT revealed that altering tyrosine 627 (Y627A/S/F) abolished GnT regulation of RIG-I/TBK1-directed IRF3 phosphorylation and transcriptional responses of ISRE, κB, and IFN-β promoters. Moreover, GnTs from NY-1V, ANDV, and TULV, but not PHV, inhibited RIG-I-directed IRF3 phosphorylation. Collectively, these findings suggest a novel role for GnTs in regulating RIG-I/TBK1 pathway-directed IRF3 phosphorylation and IFN-β induction and define virulence determinants within GnTs that may permit the attenuation of pathogenic hantaviruses. IMPORTANCE These findings provide a mechanism for selected hantavirus GnT interactions to regulate RIG-I/TBK1 signaling responses required for IFN-β induction by inhibiting TBK1 phosphorylation of IRF3. These studies culminate in showing that a single GnT residue, Y627, is required for the NY-1V GnT to inhibit RIG-I/TBK1-directed IRF3 phosphorylation and IFN-β induction. These findings define a potential virulence determinant within the NY-1V GnT that may permit hantavirus attenuation.
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Valery S
authors.initials: VS
authors.lastname: Matthys
authors.forename: Velasco
authors.initials: V
authors.lastname: Cimica
authors.forename: Nadine A
authors.initials: NA
authors.lastname: Dalrymple
authors.forename: Nicole B
authors.initials: NB
authors.lastname: Glennon
authors.forename: Chris
authors.initials: C
authors.lastname: Bianco
authors.forename: Erich R
authors.initials: ER
authors.lastname: Mackow
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral / Q000276:immunology*; D057809:HEK293 Cells; D006476:Orthohantavirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D016899:Interferon-beta / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016296:Mutagenesis; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D048008:TNF Receptor-Associated Factor 3 / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; C437968:PLAAT4 protein, human; D018168:Receptors, Retinoic Acid; D048008:TNF Receptor-Associated Factor 3; D014759:Viral Envelope Proteins; D016899:Interferon-beta; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human