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Journal of Virology, June 2006, p. 5168-5178, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02199-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

Washington B. Cárdenas,1 Yueh-Ming Loo,2 Michael Gale Jr.,2 Amy L. Hartman,3 Christopher R. Kimberlin,4 Luis Martínez-Sobrido,1 Erica Ollmann Saphire,4 and Christopher F. Basler1*

Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029,1 Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas,2 Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G-14, Atlanta, Georgia 30329,3 Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, California 920374

Received 19 October 2005/ Accepted 11 March 2006

The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-{alpha}/ß) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) · poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) · poly(rC), poly(rA) · poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-ß promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKK{varepsilon} and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

* Corresponding author. Mailing address: Department of Microbiology, Box 1124, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-4847. Fax: (212) 534-1684. E-mail: chris.basler{at}mssm.edu.

Journal of Virology, June 2006, p. 5168-5178, Vol. 80, No. 11
0022-538X/06/$08.00+0     doi:10.1128/JVI.02199-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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