Conserved Surface Features Form the Double-stranded RNA Binding Site of Non-structural Protein 1 (NS1) from Influenza A and B Viruses*

Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1–73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1–73) using NMR methods and describe the 2.1-Å x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15–93). These results identify conserved dsRNA binding surfaces on both NS1A-(1–73) and NS1B-(15–93) that are very different from those indicated in earlier “working models” of the complex between dsRNA and NS1A-(1–73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an ∼45° angle relative to the axes of helices α2/α2′. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.

[1]  R. Krug,et al.  Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs. , 1998, Molecular cell.

[2]  Tal Pupko,et al.  Structural Genomics , 2005 .

[3]  A. Nizam,et al.  Containing Pandemic Influenza at the Source , 2005, Science.

[4]  R. Krug,et al.  The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: Inhibiting the 2'-5' oligo (A) synthetase/RNase L pathway. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[5]  R. Krug,et al.  Influenza virus virulence and its molecular determinants. , 2005, Advances in virus research.

[6]  Thomas C Terwilliger,et al.  SOLVE and RESOLVE: automated structure solution and density modification. , 2003, Methods in enzymology.

[7]  J. Taubenberger,et al.  The 1918 Spanish influenza: integrating history and biology. , 2001, Microbes and infection.

[8]  M. Katze,et al.  Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor. , 1995, Virology.

[9]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[10]  T. Wolff,et al.  Double-Stranded RNA Binding of Influenza B Virus Nonstructural NS1 Protein Inhibits Protein Kinase R but Is Not Essential To Antagonize Production of Alpha/Beta Interferon , 2006, Journal of Virology.

[11]  R. Krug,et al.  Cellular antiviral responses against influenza A virus are countered at the posttranscriptional level by the viral NS1A protein via its binding to a cellular protein required for the 3' end processing of cellular pre-mRNAS. , 2003, Virology.

[12]  Hideo Goto,et al.  Avian flu: Isolation of drug-resistant H5N1 virus , 2005, Nature.

[13]  G. Montelione,et al.  A novel RNA-binding motif in influenza A virus non-structural protein 1 , 1997, Nature Structural Biology.

[14]  D. Cummings,et al.  Strategies for containing an emerging influenza pandemic in Southeast Asia , 2005, Nature.

[15]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[16]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[17]  G T Montelione,et al.  An amino-terminal polypeptide fragment of the influenza virus NS1 protein possesses specific RNA-binding activity and largely helical backbone structure. , 1995, RNA.

[18]  Helen M. Berman,et al.  Crystal structure of the unique RNA-binding domain of the influenza virus NS1 protein , 1997, Nature Structural Biology.

[19]  James M Aramini,et al.  Biophysical characterization of the complex between double-stranded RNA and the N-terminal domain of the NS1 protein from influenza A virus: evidence for a novel RNA-binding mode. , 2004, Biochemistry.

[20]  R. Fukuda,et al.  Binding of influenza A virus NS1 protein to dsRNA in vitro. , 1992, The Journal of general virology.

[21]  Prasert Auewarakul,et al.  Molecular characterization of the complete genome of human influenza H5N1 virus isolates from Thailand. , 2005, The Journal of general virology.

[22]  A. García-Sastre,et al.  Inhibition of interferon-mediated antiviral responses by influenza A viruses and other negative-strand RNA viruses. , 2001, Virology.

[23]  Mark Gerstein,et al.  Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. , 2005, Methods in enzymology.

[24]  R. Krug,et al.  Influenza A virus NS1 protein targetspoly(A)‐binding protein II of the cellular 3′‐end processing machinery , 1999, The EMBO journal.

[25]  김삼묘,et al.  “Bioinformatics” 특집을 내면서 , 2000 .

[26]  R. Krug,et al.  Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)‐induced ubiquitin‐like ISG15 protein , 2001, The EMBO journal.

[27]  R. Krug,et al.  Intracellular warfare between human influenza viruses and human cells: the roles of the viral NS1 protein. , 2003, Virology.

[28]  V. Georgiev.  Virology , 1955, Nature.

[29]  C. Naeve,et al.  Large-Scale Sequence Analysis of Avian Influenza Isolates , 2006, Science.

[30]  James M Aramini,et al.  SPINS: A laboratory information management system for organizing and archiving intermediate and final results from NMR protein structure determinations , 2006, Proteins.

[31]  A. García-Sastre,et al.  A Recombinant Influenza A Virus Expressing anRNA-Binding-Defective NS1 Protein Induces High Levels of BetaInterferon and Is Attenuated inMice , 2003, Journal of Virology.

[32]  G. Montelione,et al.  Structural basis for ubiquitin-like ISG 15 protein binding to the NS1 protein of influenza B virus: a protein-protein interaction function that is not shared by the corresponding N-terminal domain of the NS1 protein of influenza A virus. , 2002, Virology.

[33]  Yi Guan,et al.  Oseltamivir resistance during treatment of influenza A (H5N1) infection. , 2005, The New England journal of medicine.

[34]  Yoshihiro Kawaoka,et al.  Influenza: lessons from past pandemics, warnings from current incidents , 2005, Nature Reviews Microbiology.

[35]  A. García-Sastre,et al.  The N- and C-Terminal Domains of the NS1 Protein of Influenza B Virus Can Independently Inhibit IRF-3 and Beta Interferon Promoter Activation , 2004, Journal of Virology.

[36]  C. Dominguez,et al.  HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. , 2003, Journal of the American Chemical Society.

[37]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[38]  R. Krug,et al.  The RNA-binding and effector domains of the viral NS1 protein are conserved to different extents among influenza A and B viruses. , 1996, Virology.

[39]  G. Montelione,et al.  RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids. , 1999, RNA.

[40]  R. Rosenfeld.  Nature , 2009, Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery.