PMID:16698997

Citation	Cárdenas, WB, Loo, YM, Gale, M Jr, Hartman, AL, Kimberlin, CR, Martínez-Sobrido, L, Saphire, EO and Basler, CF (2006) Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling. J. Virol. 80:5168-78
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.
Links	PubMed PMC1472134 Online version:10.1128/JVI.02199-05
Keywords	Animals; Cell Line; Cercopithecus aethiops; DEAD-box RNA Helicases/physiology; Ebolavirus/chemistry; Ebolavirus/metabolism; Humans; Interferon-alpha/metabolism; Interferon-beta/metabolism; RNA, Double-Stranded/drug effects; RNA, Double-Stranded/metabolism; Signal Transduction/drug effects; Signal Transduction/physiology; Vero Cells; Viral Proteins/pharmacology; Viral Regulatory and Accessory Proteins
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
EBOZM:VP35	involved_in	GO:0039501: suppression by virus of host type I interferon production	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
EBOZM:VP35		GO:0003690: double-stranded DNA binding	ECO:0000315:		F		Figure 1. All of these results show VP35s ability to bind dsDNA. An in vitro "pull-down" assay was used. Cell lysates were prepared and incubated with pIC-Sepharose and then visualized by Western blotting.	complete CACAO 6914
EBOZM:VP35		GO:0039501: suppression by virus of host type I interferon production	ECO:0000315:		P		Figure 2. The abilities of wild-type VP35, R312A, and K309A to inhibit the SeV-induced activation of an IFN-Beta promoter reporter plasmid were compared. Western blotting was used to demonstrate the ability of VP35 to regulate IFN-Beta in Figure 2A, while Figure B shows the results from a panel overlay onto Vero cells in a 96-well plate and subsequent fluorescence.	complete CACAO 6915

References

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