title: E339...R416 salt bridge of nucleoprotein as a feasible target for influenza virus inhibitors.
abstract: The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism--disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozin-resistant strains. This work provides a new approach to design antiinfluenza drugs.
authors.affiliation: Institute of Biological Chemistry and  Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yu-Fang
authors.initials: YF
authors.lastname: Shen
authors.forename: Yu-Hou
authors.initials: YH
authors.lastname: Chen
authors.forename: Shao-Ying
authors.initials: SY
authors.lastname: Chu
authors.forename: Meng-I
authors.initials: MI
authors.lastname: Lin
authors.forename: Hua-Ting
authors.initials: HT
authors.lastname: Hsu
authors.forename: Pei-Yu
authors.initials: PY
authors.lastname: Wu
authors.forename: Chao-Jung
authors.initials: CJ
authors.lastname: Wu
authors.forename: Hui-Wen
authors.initials: HW
authors.lastname: Liu
authors.forename: Fu-Yang
authors.initials: FY
authors.lastname: Lin
authors.forename: Gialih
authors.initials: G
authors.lastname: Lin
authors.forename: Pang-Hung
authors.initials: PH
authors.lastname: Hsu
authors.forename: An-Suei
authors.initials: AS
authors.lastname: Yang
authors.forename: Yih-Shyun E
authors.initials: YS
authors.lastname: Cheng
authors.forename: Ying-Ta
authors.initials: YT
authors.lastname: Wu
authors.forename: Chi-Huey
authors.initials: CH
authors.lastname: Wong
authors.forename: Ming-Daw
authors.initials: MD
authors.lastname: Tsai
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004285:Dogs; D016503:Drug Delivery Systems / Q000379:methods; D019084:Fluorescent Antibody Technique, Indirect; D006860:Hydrogen Bonding; D008156:Luciferases; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense / Q000235:genetics; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism*; D009975:Orthomyxoviridae / Q000235:genetics*; D011487:Protein Conformation; D055503:Protein Multimerization; D055672:Static Electricity; D014461:Ultracentrifugation; D014779:Virus Replication / Q000235:genetics*
chemical_list: D017931:DNA Primers; D046912:Multiprotein Complexes; D009698:Nucleoproteins; D008156:Luciferases