Structure of the Ebola VP35 interferon inhibitory domain

Abstract

Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 A and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.

Fig. 1.

Crystal structure of VP35 C-terminal IFN-inhibitory domain (IID) reveals a fold that binds dsRNA. (A) Domain organization of VP35. (B) Ribbon representation of VP35 IID. Secondary structural elements that form the α-helical subdomain (orange) and the β-sheet subdomain (yellow). (C) Topology and delimiting sequence markers of VP35 IID.

Fig. 2.

The surface area between the VP35 IID subdomains is hydrophobic. (A and B) Electrostatic representations of the intersubdomain interaction surface for the α-helical subdomain (A) and the β-sheet subdomain (B) reveal hydrophobic surfaces buried between the 2 subdomains. Red, white, and blue represent negative, neutral, and positive electrostatic potentials, respectively (range −5 to +5 kT). (C) Stereographic image showing the Trp-324 side chain making important hydrophobic contacts with residues in β4 strand, α5 helix, and PPII. The 2F_o−F_c map is contoured at 1σ (blue) and 2σ (pink).

Fig. 3.

Conserved basic residues in the β-sheet domain form an extended cluster. (A) Electrostatic representation of the solvent-exposed surface of VP35 IID. (B) Conserved basic residues important for IFN antagonism and dsRNA binding are highlighted.

Fig. 4.

Mutation of conserved basic residues in the β-sheet subdomain diminishes dsRNA binding. (A) Native gel-shift assays show that wild-type VP35 IID and not basic patch mutants bind dsRNA with high affinity. Lanes: M, marker; 1, dsRNA alone; 2, dsRNA plus wild-type VP35 IID; 3, dsRNA plus VP35 IID Lys309Ala; 4, dsRNA plus VP35 IID Arg312Ala; 5, dsRNA plus VP35 IID Lys319Ala; and 6, dsRNA plus VP35 IID Lys339Ala. Coomassie-stained SDS/PAGE gel highlights the purity and the relative amounts of VP35 IID proteins. (B) ¹H/¹⁵N HSQC NMR-spectra of wild-type and mutant VP35 IID proteins show localized chemical shift changes (red circles).

Fig. 5.

The RNA-binding domains of Ebola VP35 and influenza NS1 proteins are incorporated into distinct scaffolds. (A and B) Structures of the Ebola VP35 IID (this study; left) (A) and the influenza NS1A dsRNA binding domain (PDB ID code 2ZKO) (B). (C) Similar side chains of arginine and lysine residues in the 8-residue alignment between the NS1A and VP35 protein sequences are shown and highlighted in gray (adapted from ref. 21).

Fig. 6.

Model for VP35-mediated IFN inhibition and immune suppression.