title: The RNA-binding domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner.
abstract: Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs. NS1 is known to bind non-specifically to double-stranded RNA (dsRNA) as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs. Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5' terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide sequence located 15 nucleotides downstream from NS1's stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5'-GUAAC / 3'-CUUAG double-stranded motif, which closely resembles the canonical 5'-splice site. Through an in-depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1's RNA-binding domain (RBD) specifically recognizes sequence patterns in a structure-dependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar K(D) values) that leads to oligomerization of the RBD on its RNA ligands.
authors.affiliation: Equipe BioVA, UMR1282 Infectiologie et Santé Publique, Institut National de la Recherche Agronomique, Nouzilly F-37380, France. daniel.marc@tours.inra.fr
authors.forename: Daniel
authors.initials: D
authors.lastname: Marc
authors.forename: Sosthène
authors.initials: S
authors.lastname: Barbachou
authors.forename: Denis
authors.initials: D
authors.lastname: Soubieux
mesh_terms: D019943:Amino Acid Substitution; D052157:Aptamers, Nucleotide / Q000737:chemistry; D009980:Influenza A virus / Q000235:genetics*; D059372:Nucleotide Motifs; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; C085820:INS1 protein, influenza virus; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017361:Viral Nonstructural Proteins