Summary The VP35 protein encoded by the highly pathogenic Ebola virus facilitates immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I like receptors. Here we report the crystal structure of Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which reveals how VP35-dsRNA interactions contribute to immune evasion, and corresponding in vivo studies. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are “end-capped” by a pocket of hydrophobic residues that mimics blunt end dsRNA recognition by RIG-I-like receptors. Residues that are critical for RNA binding are also important for interferon inhibition in vivo, but not for viral polymerase co-factor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.

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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...

The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-␣/␤) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

Ebola viruses (EBOV) are causative agents of lethal hemorrhagic fever in humans and non-human primates. Among the filoviruses characterized thus far, Reston Ebola virus (REBOV) is the only Ebola species that is non-pathogenic in humans despite the fact that REBOV can cause lethal disease in non-human primates. Previous studies also suggest that Reston EBOV is less effective at inhibiting host innate immune responses, compared with Zaire EBOV or Marburg virus. Virally encoded VP35 protein is critical for immune suppression, but an understanding of the relative contributions of VP35 proteins from REBOV and other filoviruses is currently lacking. In order to address this question, we characterized REBOV VP35 IFN inhibitory domain (IID) using structural, biochemical, and virological studies. These studies reveal differences in dsRNA binding and IFN inhibition between the two species. These observed differences are likely due to increased stability and loss of flexibility in REBOV VP35 IID as demonstrated by thermal-shift stability assays. Consistent with this finding, our 1.71 Å crystal structure of the REBOV VP35 IID reveal that the structure is highly similar to ZEBOV VP35 IID with an overall backbone r.m.s.d. of 0.64 Å, but contains an additional helical element at the linker between the two subdomains of VP35 IID. Mutations near the linker, including swapping sequences between REBOV and ZEBOV, Corresponding author: Gaya K. Amarasinghe, Tel: (515) Fax: (515) 294-0453, amarasin@iastate.edu. 7 equal contribution Accession numbers: Coordinates and structure factors for Reston Ebola VP35 IID protein has been deposited in the Protein Data Bank under the PDB ID: 3L2A.Note added during manuscript preparation: While this manuscript was in preparation, a structure of the Reston Ebola VP35 IID free and bound to 18 bp dsRNA has appeared online and based on the published images, the two structures appear similar 48 . However, we were unable to compare as the coordinates for these structures were not released at the time of initial submission.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 19; 20 . However, we and others have shown that VP35 binds dsRNA through the Cterminal IID, which alone is sufficient for interferon (IFN) inhibition 15; 20; 21; 22 . Our recent crystal structure of the ZEBOV VP35 IID revealed a cluster of conserved basic residues that are important for dsRNA binding 27 . From this first structure of a VP35 IID and associated solution NMR experiments, we demonstrated that VP35 IID consists of two subdomains that form a single independently folded unit....