Mutations abrogating VP35 interaction with double-stranded RNA render Ebola virus avirulent in guinea pigs.
Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-alpha/beta 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-alpha/beta 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.
Prins
Kathleen C
KC
Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Delpeut
Sebastien
S
Leung
Daisy W
DW
Reynard
Olivier
O
Volchkova
Valentina A
VA
Reid
St Patrick
SP
Ramanan
Parameshwaran
P
Cárdenas
Washington B
WB
Amarasinghe
Gaya K
GK
Volchkov
Viktor E
VE
Basler
Christopher F
CF
D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D029043:Ebolavirus / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D005260:Female; D006168:Guinea Pigs / Q000821:virology*; D006801:Humans; D007155:Immunologic Factors / Q000235:genetics / Q000276:immunology; D016898:Interferon-alpha / Q000235:genetics / Q000276:immunology; D016899:Interferon-beta / Q000235:genetics / Q000276:immunology; D007372:Interferons / Q000037:antagonists & inhibitors / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014709:Vero Cells; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007155:Immunologic Factors; D016898:Interferon-alpha; D012330:RNA, Double-Stranded; C080980:VP35 protein, filovirus; D054334:Viral Regulatory and Accessory Proteins; D016899:Interferon-beta; D007372:Interferons