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Journal of Virology, September 2007, p. 8967-8976, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.02523-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Ebola Virus VP30 Is an RNA Binding Protein

Sinu P. John,¹ Tan Wang,² Scott Steffen,^3, Sonia Longhi,⁴ Connie S. Schmaljohn,³ and Colleen B. Jonsson^2*

Graduate Program in Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294,¹ Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, Alabama 35205,² U.S. Army Medical Research Institute of Infectious Diseases, Ft. Detrick, Maryland 21702,³ Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS, and Universités Aix-Marseille I et II, Campus de Luminy, 13288 Marseille Cedex 09, France⁴

Received 15 November 2006/ Accepted 31 May 2007

The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn²⁺, but not Mg²⁺, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

Published ahead of print on 13 June 2007.

Present address: Celera Genomics, Department of Protein Therapeutics, 45 West Gude Dr., Rockville, MD 20850.