title: Ebola virus VP30 is an RNA binding protein.
abstract: 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. Zn(2+), but not Mg(2+), 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.
authors.affiliation: Graduate Program in Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
authors.forename: Sinu P
authors.initials: SP
authors.lastname: John
authors.forename: Tan
authors.initials: T
authors.lastname: Wang
authors.forename: Scott
authors.initials: S
authors.lastname: Steffen
authors.forename: Sonia
authors.initials: S
authors.lastname: Longhi
authors.forename: Connie S
authors.initials: CS
authors.lastname: Schmaljohn
authors.forename: Colleen B
authors.initials: CB
authors.lastname: Jonsson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003067:Coenzymes / Q000494:pharmacology; D019295:Computational Biology; D029043:Ebolavirus / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012330:RNA, Double-Stranded / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017384:Sequence Deletion; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
chemical_list: D003067:Coenzymes; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014157:Transcription Factors; C479897:VP30 protein, ebola virus; D014764:Viral Proteins; D008274:Magnesium; D015032:Zinc