Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A Virus

ABSTRACT The retinoic acid-inducible gene I product (RIG-I) has been identified as a cellular sensor of RNA virus infection resulting in beta interferon (IFN-β) induction. However, many viruses are known to encode viral products that inhibit IFN-β production. In the case of influenza A virus, the viral nonstructural protein 1 (NS1) prevents the induction of the IFN-β promoter by inhibiting the activation of transcription factors, including IRF-3, involved in IFN-β transcriptional activation. The inhibitory properties of NS1 appear to be due at least in part to its binding to double-stranded RNA (dsRNA), resulting in the sequestration of this viral mediator of RIG-I activation. However, the precise effects of NS1 on the RIG-I-mediated induction of IFN-β have not been characterized. We now report that the NS1 of influenza A virus interacts with RIG-I and inhibits the RIG-I-mediated induction of IFN-β. This inhibition was apparent even when a mutant RIG-I that is constitutively activated (in the absence of dsRNA) was used to trigger IFN-β production. Coexpression of RIG-I, its downstream signaling partner, IPS-1, and NS1 resulted in increased levels of RIG-I and NS1 within an IPS-1-rich, solubilization-resistant fraction after cell lysis. These results suggest that RIG-I, IPS-1, and NS1 become part of the same complex. Consistent with this idea, NS1 was also found to inhibit IFN-β promoter activation by IPS-1 overexpression. Our results indicate that, in addition to sequestering dsRNA, the NS1 of influenza A virus binds to RIG-I and inhibits downstream activation of IRF-3, preventing the transcriptional induction of IFN-β.

[1]  S. Goodbourn,et al.  The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFN-beta promoter. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[2]  G. Kochs,et al.  The interferon response circuit: Induction and suppression by pathogenic viruses , 2005, Virology.

[3]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[4]  A. García-Sastre,et al.  Engineered viral vaccine constructs with dual specificity: avian influenza and Newcastle disease. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Z. Zhai,et al.  VISA is an adapter protein required for virus-triggered IFN-beta signaling. , 2005, Molecular cell.

[6]  Osamu Takeuchi,et al.  Cell type-specific involvement of RIG-I in antiviral response. , 2005, Immunity.

[7]  H. Klenk,et al.  The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3 , 2003, Journal of Virology.

[8]  Nicole M. Bouvier,et al.  Newcastle Disease Virus (NDV)-Based Assay Demonstrates Interferon-Antagonist Activity for the NDV V Protein and the Nipah Virus V, W, and C Proteins , 2003, Journal of Virology.

[9]  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.

[10]  Shizuo Akira,et al.  Innate immune recognition of viral infection , 2006, Nature Immunology.

[11]  R. Webby,et al.  Mutations in the NS1 Protein of Swine Influenza Virus Impair Anti-Interferon Activity and Confer Attenuation in Pigs , 2005, Journal of Virology.

[12]  R. Tsien,et al.  A monomeric red fluorescent protein , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  H. Bluyssen,et al.  The interferon-stimulated gene 54 K promoter contains two adjacent functional interferon-stimulated response elements of different strength, which act synergistically for maximal interferon-alpha inducibility. , 1994, European journal of biochemistry.

[14]  Adolfo Garcia-Sastre,et al.  Influenza Virus NS1 Protein Counteracts PKR-Mediated Inhibition of Replication , 2000, Journal of Virology.

[15]  H. Niwa,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector. , 1991, Gene.

[16]  F. Weber,et al.  Activation of Innate Defense against a Paramyxovirus Is Mediated by RIG-I and TLR7 and TLR8 in a Cell-Type-Specific Manner , 2005, Journal of Virology.

[17]  J. Hiscott,et al.  MasterCARD: a priceless link to innate immunity. , 2006, Trends in molecular medicine.

[18]  R. Flavell,et al.  TLR-Independent Induction of Dendritic Cell Maturation and Adaptive Immunity by Negative-Strand RNA Viruses1 , 2004, The Journal of Immunology.

[19]  Osamu Takeuchi,et al.  IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction , 2005, Nature Immunology.

[20]  B. Prasad,et al.  X-ray structure of influenza virus NS1 effector domain , 2006, Nature Structural &Molecular Biology.

[21]  M. Katze,et al.  Global Host Immune Response: Pathogenesis and Transcriptional Profiling of Type A Influenza Viruses Expressing the Hemagglutinin and Neuraminidase Genes from the 1918 Pandemic Virus , 2004, Journal of Virology.

[22]  Robert H. Silverman,et al.  Functional Replacement of the Carboxy-Terminal Two-Thirds of the Influenza A Virus NS1 Protein with Short Heterologous Dimerization Domains , 2002, Journal of Virology.

[23]  A. García-Sastre,et al.  Activation of Interferon Regulatory Factor 3 Is Inhibited by the Influenza A Virus NS1 Protein , 2000, Journal of Virology.

[24]  K. Ishii,et al.  Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses , 2006, Nature.

[25]  Shizuo Akira,et al.  Toll-like receptor signalling , 2004, Nature Reviews Immunology.

[26]  S. Elankumaran,et al.  Recombinant Newcastle disease virus as a vaccine vector. , 2003, Poultry science.

[27]  Zhijian J. Chen,et al.  Identification and Characterization of MAVS, a Mitochondrial Antiviral Signaling Protein that Activates NF-κB and IRF3 , 2005, Cell.

[28]  A. García-Sastre,et al.  Attenuation of Equine Influenza Viruses through Truncations of the NS1 Protein , 2005, Journal of Virology.

[29]  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.

[30]  Thorsten Wolff,et al.  The Influenza A Virus NS1 Protein Inhibits Activation of Jun N-Terminal Kinase and AP-1 Transcription Factors , 2002, Journal of Virology.

[31]  S. Lemon,et al.  Regulating Intracellular Antiviral Defense and Permissiveness to Hepatitis C Virus RNA Replication through a Cellular RNA Helicase, RIG-I , 2005, Journal of Virology.

[32]  A. García-Sastre,et al.  Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[33]  H. Katinger,et al.  Immunogenicity and Protection Efficacy of Replication-Deficient Influenza A Viruses with Altered NS1 Genes , 2004, Journal of Virology.

[34]  K. Tyler,et al.  Does Toll-like receptor 3 play a biological role in virus infections? , 2004, Virology.

[35]  M. Brunda,et al.  Interferons and Other Cytokines , 1997 .

[36]  R. Krug,et al.  Binding of the influenza A virus NS1 protein to PKR mediates the inhibition of its activation by either PACT or double-stranded RNA. , 2006, Virology.

[37]  D. Levy,et al.  Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. , 1998, Virology.

[38]  G. Stark,et al.  How cells respond to interferons. , 1998, Annual review of biochemistry.

[39]  A. García-Sastre,et al.  Recombinant Newcastle Disease Virus as a Vaccine Vector , 2001, Journal of Virology.

[40]  Z. Zhai,et al.  VISA Is an Adapter Protein Required for Virus-Triggered IFN-β Signaling , 2005 .

[41]  S. Akira,et al.  Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8 , 2004, Science.

[42]  D. Levy,et al.  IRF3 and IRF7 Phosphorylation in Virus-infected Cells Does Not Require Double-stranded RNA-dependent Protein Kinase R or IκB Kinase but Is Blocked by Vaccinia Virus E3L Protein* , 2001, The Journal of Biological Chemistry.

[43]  William M. Lee,et al.  Viral and therapeutic control of IFN-beta promoter stimulator 1 during hepatitis C virus infection. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Christine A. Biron,et al.  Type 1 Interferons and the Virus-Host Relationship: A Lesson in Détente , 2006, Science.

[45]  Shizuo Akira,et al.  The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses , 2004, Nature Immunology.

[46]  Qiang Sun,et al.  Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKε Molecular Complex from the Mitochondrial Outer Membrane by Hepatitis C Virus NS3-4A Proteolytic Cleavage , 2006, Journal of Virology.

[47]  Shizuo Akira,et al.  Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA , 2004, Science.

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

[49]  Amer A. Beg,et al.  Influenza A Virus NS1 Protein Prevents Activation of NF-κB and Induction of Alpha/Beta Interferon , 2000, Journal of Virology.

[50]  F. Hayden,et al.  Symptom pathogenesis during acute influenza: Interleukin‐6 and Other cytokine responses , 2001, Journal of medical virology.

[51]  R. Flavell,et al.  Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3 , 2001, Nature.

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

[53]  A. Bowie,et al.  Viral Activation of Macrophages through TLR-Dependent and -Independent Pathways1 , 2004, The Journal of Immunology.

[54]  M. Severa,et al.  Tumor Necrosis Factor Alpha Enhances Influenza A Virus-Induced Expression of Antiviral Cytokines by Activating RIG-I Gene Expression , 2006, Journal of Virology.

[55]  Ralf Bartenschlager,et al.  Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus , 2005, Nature.

[56]  Akiko Iwasaki,et al.  Recognition of single-stranded RNA viruses by Toll-like receptor 7. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[57]  F. Weber,et al.  Double-Stranded RNA Is Produced by Positive-Strand RNA Viruses and DNA Viruses but Not in Detectable Amounts by Negative-Strand RNA Viruses , 2006, Journal of Virology.

[58]  Roger E Bumgarner,et al.  Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza , 2002, Proceedings of the National Academy of Sciences of the United States of America.