Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

Abstract

Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-␣/␤ responses that also functions as a viral polymerase cofactor.Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-␣/␤ production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.Ebola viruses (EBOVs) are zoonotic, enveloped negativestrand RNA viruses belonging to the family Filoviridae which cause lethal viral hemorrhagic fever in humans and nonhuman primates (47). Currently, information regarding EBOV-encoded virulence determinants remains limited. This, coupled with our lack of understanding of biochemical and structural properties of virulence factors, limits efforts to develop novel prophylactic or therapeutic approaches toward these infections.It has been proposed that EBOV-encoded mechanisms to counter innate immune responses, particularly interferon (IFN) responses, are critical to EBOV pathogenesis (7). However, a role for viral immune evasion functions in the pathogenesis of lethal EBOV infection has yet to be demonstrated.Of the eight major EBOV gene products, two viral proteins have been demonstrated to counter host IFN responses. The VP35 protein is a viral polymerase cofactor and structural protein that also inhibits IFN-␣/␤ production by preventing the activation of interferon regulatory factor (IRF)- 3 and -7 (3, 4, 8, 24, 27, 34, 41). VP35 also inhibits the activation of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated kinase with antiviral activity, and inhibits RNA silencing (17,20,48). The VP24 protein is a minor structural protein implicated in virus assembly and regulation of viral RNA synthesis, and changes in VP24 coding sequences are also associated with adaptation of EBOVs to mice and guinea pigs (2,13,14,27,32,37,50,52). Further, VP24 inhibits cellular responses to both IFN-␣/␤ an...

Structural basis for Marburg virus VP35–mediated immune evasion mechanisms

“…Although the nature of the exact ligands that activate RLRs in vivo have not yet been definitively identified, dsRNA-bound RIG-I and MDA5 structures show that multiple PAMPs, including double-strandedness, dsRNA blunt ends, and short dsRNA with 5′OH or 5′PPP, can activate RLRs (34). A correlation between dsRNA-binding ability of zVP35 and its IFN-inhibitory effect on RLR function has been documented in several studies (15,16,19,21), and its biological relevance is supported by the observations that preactivation of RIG-I dramatically decreases ZEBOV yield and that recombinant ZEBOVs with mutations at critical RNAbinding residues are attenuated in vitro and avirulent in vivo (29,41). Our structural findings are consistent with the in vitro dsRNA-binding studies, suggesting that the crystal structure reflects a potentially physiologically relevant complex.…”

“…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). Consistent with this, mutation of CBP residues abrogates the dsRNA-binding and IFN-inhibitory activities of zVP35 and greatly attenuated virus replication in IFN-competent cells and in vivo (15,21,27,29). These studies also demonstrated that zIID/rIID proteins end cap dsRNA, potentially shielding this blunt-end dsRNA pathogen-associated molecular pattern (PAMP) from detection by RLRs (21).…”

Post-exposure treatments for Ebola and Marburg virus infections

“…Some of the first cells to be infected are immune surveillance cells, including dendritic cells, monocytes and macrophages, in which the infection-mediated blockade of immune activation is particularly powerful as it reduces the potential for these cells to become activated early in the course of infection. The contribution of VP35 to immune evasion is best exhibited in vivo: Ebolavirus with mutant VP35 is non-lethal in animal models including mice 108 and guinea pigs 109 .…”

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.…”