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-␣/␤) 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 vitrotranscribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or doublestranded 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-␤ promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKK 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.

Predictive and comparative analysis of Ebolavirus proteins

“…Host immune suppression by VP35 is mainly related to its interaction with dsRNA, but the loss of dsRNA-binding ability does not completely abolish VP35-mediated immune suppression. 96 This observation indicates the existence of other mechanisms for immune suppression by VP35, where the surface enriched in RESTV-specific mutations may play a role. One RESTV-specific mutation (T226A) is adjacent to the position in VP24 that is mutated (T50I) during adaptation to mice 97,98 (orange circles in Fig.…”

Filovirus Replication and Transcription

“…It is already known for the polymerase cofactor VP35 that it interferes with the innate immune system [26][27][28]. Within the same vein, bioinformatic analysis has revealed that filovirus species differing in their replication efficiency also differ in their ability to evade the cellular antiviral response [71].…”

“…Additional functions have been determined for VP35, which has been shown to act as a type I interferon antagonist by interfering with retinoic acid inducible gene I-dependent activation of the interferon regulatory factor 3 and to block the activation of the double-stranded RNAdependent protein kinase [26][27][28].…”

Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

“…In fact, Ebola possesses several strategies to antagonize IFN production and signaling. Ebola VP35 prevents IFN production by blocking activation of IFN regulatory factor 3, and VP24 inhibits IFN responsiveness by blocking the nuclear accumulation of tyrosine-phosphorylated STAT1 (20,21). These proteins likely suppress de novo IFN-induced synthesis of viral restriction factors, but constitutive expression in various cell types remains a restrictive barrier to viral infection.…”