Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding

The influenza virus nucleoprotein (NP) is a single-strand-RNA-binding prote in associated with genome and antigenome RNA and is one of the four virus p roteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by which NP binds RNA, we undertook a physical and mutational analysis of the polypeptide, with the strategy of identifyin g first the regions in direct contact with RNA, then the classes of amino a cids involved, and finally the crucial residues by mutagenesis. Chemical fr agmentation and amino acid sequencing of NP that had been UV cross linked t o radiolabelled RNA showed that protein-RNA contacts occur throughout the l ength of the polypeptide. Chemical modification experiments implicated argi nine but not lysine residues as important for RNA binding, while RNA-depend ent changes in the intrinsic fluorescence spectrum of NP suggested the invo lvement of tryptophan residues. Supporting these observations, single-codon mutagenesis identified five tryptophan, one phenylalanine, and two arginin e residues as essential for high-affinity RNA binding at physiological temp erature. In addition, mutants unable to bind RNA in vitro were unable to su pport virus gene expression in vivo. The mutationally sensitive residues ar e not localized to any particular region of NP but instead are distributed throughout the protein. Overall, these data are inconsistent with previous models suggesting that the NP-RNA interaction is mediated by a discrete N-t erminal domain. Instead, we propose that high-affinity binding of RNA by NP requires the concerted interaction of multiple regions of the protein with RNA and that the individual protein-RNA contacts are mediated by a combina tion of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases.