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 Å 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.crystal structure ͉ Ebola virus ͉ RNA binding E bola viruses (EBOVs) cause severe hemorrhagic fever characterized by fever, shock, coagulation defects, and impaired immunity (1, 2). These manifestations of infection are thought to reflect subversion of the innate immune system coupled with uncontrolled viral replication, particularly in macrophages and dendritic cells (3,4). EBOV infection of these cells enhances production of proinflammatory cytokines, such as TNF-␣ and IFN (IFN)-␥, and diminishes stimulation of T cell maturation by dendritic cells (3, 4). Like other negative-strand RNA viruses that impair both innate and adaptive immunity (e.g., influenza, rabies, and measles), EBOV suppresses host IFN activities to replicate, thus resulting in serious disease (5, 6). Only individuals who survive EBOV infection show appreciable amounts of viral-specific antibodies (7), suggesting that EBOV infections lead to shutdown of early immune responses and prevent activation of adaptive immune responses.Recognition of viral particles and viral RNA, including RNA modifications such as 5Ј-triphosphate (5Ј-ppp), by cytosolic pattern recognition receptor helicases RIG-I and MDA-5 leads to activation of transcription factors, including IFN regulatory factor-3 (IRF-3), IRF-7, NF-B, and AP-1 (8-12). These transcription factors in turn induce expression of a large number of cytokines, such as IFN-␣ and IFN-␤ (13). Activated IFN genes operate in both autocrine and paracrin...

Predictive and comparative analysis of Ebolavirus proteins

“…Ebolavirus proteins contain a significant fraction (20%) of structurally disordered regions, and the fraction of variable positions in these regions is significantly higher (p < 0.01) than in the structurally ordered regions. The 3D structures of globular regions are mostly known (Table S2) [54][55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71] except for the N-terminal zinc-finger domain of VP30, the coiled-coil domain of VP35, and protein L. Identification and analysis of structurally characterized homologs allowed us to predict the structure of the zinc-finger domain in VP30, the overall topology of NP, and the structure and catalytic sites for the catalytic domains of protein L.…”

Molecular dynamics exploration of the binding mechanism and properties of single-walled carbon nanotube to WT and mutant VP35 FBP region of Ebola virus

“…The crystal structures of WT VP35 (PDB ID: 3FKE), which is a dipolymer that just contains the C-terminal domain of VP35, were obtained from the Protein Data Bank [15]. All of the crystal water molecules were deleted from the PDB file.…”

“…VP35 consists in an N-terminal coiled-coil domain and a Cterminal domain termed the IFN inhibitory domain (IID) [12][13][14]. The C-terminal domain of VP35 includes two basic patches: the first basic patch (FBP) is important for interactions with EBOV nucleoprotein (NP) and VP35 polymerase cofactor function; the central basic patch (CBP) is important for VP35-dsRNA binding and IFN inhibition [14,15]. In the absence of VP35, no reported gene expression has been detected, while in the presence of optimal concentration of VP35, maximal reported gene activation has been detected [16,17].…”

Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

“…These include inhibition of IFN production through double-stranded (ds)RNA sequestration and inhibition of IFN regulatory factor (IRF) 3/IRF7 phosphorylation by direct association with kinases that activate IRF3/IRF7, IKKe/TBK-1 (13,15,21,25). Structural and biochemical studies on Zaire EBOV (ZEBOV) and Reston EBOV (REBOV) VP35 IFN inhibitory domains (IID) (termed zVP35 and zIID or rVP35 and rIID, for VP35 protein and IID, respectively) in free and dsRNA-bound forms identified a number of functionally critical basic residues (21,(26)(27)(28). These are located in the central basic patch (CBP) in the β-sheet subdomain and the first basic patch (FBP) in the α-helical subdomain (21,(26)(27)(28).…”

“…Structural and biochemical studies on Zaire EBOV (ZEBOV) and Reston EBOV (REBOV) VP35 IFN inhibitory domains (IID) (termed zVP35 and zIID or rVP35 and rIID, for VP35 protein and IID, respectively) in free and dsRNA-bound forms identified a number of functionally critical basic residues (21,(26)(27)(28). These are located in the central basic patch (CBP) in the β-sheet subdomain and the first basic patch (FBP) in the α-helical subdomain (21,(26)(27)(28). Based on the dsRNA-bound structures of VP35 IIDs, it was suggested that these basic patches are important for protein-protein and protein-RNA interactions (21,27,28).…”