title: Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling.
abstract: The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-alpha/beta) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) . poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) . poly(rC), poly(rA) . poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-beta promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKepsilon and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA.
authors.forename: Washington B
authors.initials: WB
authors.lastname: Cárdenas
authors.forename: Yueh-Ming
authors.initials: YM
authors.lastname: Loo
authors.forename: Michael
authors.initials: M
authors.lastname: Gale
authors.forename: Amy L
authors.initials: AL
authors.lastname: Hartman
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Kimberlin
authors.forename: Luis
authors.initials: L
authors.lastname: Martínez-Sobrido
authors.forename: Erica Ollmann
authors.initials: EO
authors.lastname: Saphire
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Basler
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000502:physiology*; D029043:Ebolavirus / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000187:drug effects* / Q000378:metabolism; D011971:Receptors, Immunologic; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014709:Vero Cells; D014764:Viral Proteins / Q000494:pharmacology*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D016898:Interferon-alpha; D012330:RNA, Double-Stranded; D011971:Receptors, Immunologic; C080980:VP35 protein, filovirus; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D016899:Interferon-beta; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases