Influenza Virus Evades Innate and Adaptive Immunity via the NS1 Protein

ABSTRACT Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1β, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-α/β, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.

[1]  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.

[2]  S. Sealfon,et al.  Transcriptome Fingerprints Distinguish Hallucinogenic and Nonhallucinogenic 5-Hydroxytryptamine 2A Receptor Agonist Effects in Mouse Somatosensory Cortex , 2003, The Journal of Neuroscience.

[3]  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.

[4]  J. Hiscott,et al.  Gene Expression and Antiviral Activity of Alpha/Beta Interferons and Interleukin-29 in Virus-Infected Human Myeloid Dendritic Cells , 2005, Journal of Virology.

[5]  R. Steinman,et al.  Dendritic cells and the control of immunity , 1998, Nature.

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

[7]  J. Renauld,et al.  IL-23 and IL-12 Have Overlapping, but Distinct, Effects on Murine Dendritic Cells1 , 2002, The Journal of Immunology.

[8]  J. Connolly,et al.  Upon viral exposure, myeloid and plasmacytoid dendritic cells produce 3 waves of distinct chemokines to recruit immune effectors. , 2006, Blood.

[9]  S. Akira,et al.  Selective contribution of IFN-α/β signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  H. Katinger,et al.  Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells , 1998, Journal of Virology.

[11]  A. García-Sastre,et al.  Newcastle Disease Virus V Protein Is a Determinant of Host Range Restriction , 2003, Journal of Virology.

[12]  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.

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

[14]  A. Fernández-Sesma,et al.  Th2 responses to inactivated influenza virus can Be converted to Th1 responses and facilitate recovery from heterosubtypic virus infection. , 1999, The Journal of infectious diseases.

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

[16]  A. García-Sastre Mechanisms of inhibition of the host interferon α/β-mediated antiviral responses by viruses , 2002 .

[17]  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.

[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]  Antonio Alcami,et al.  Viral mechanisms of immune evasion , 2000, Immunology Today.

[20]  A. Fernández-Sesma,et al.  A bispecific antibody recognizing influenza A virus M2 protein redirects effector cells to inhibit virus replication in vitro , 1996, Journal of virology.

[21]  G. Trinchieri,et al.  Interleukin-12 and the regulation of innate resistance and adaptive immunity , 2003, Nature Reviews Immunology.

[22]  W G Laver,et al.  Molecular mechanisms of variation in influenza viruses , 1982, Nature.

[23]  A. García-Sastre Mechanisms of inhibition of the host interferon alpha/beta-mediated antiviral responses by viruses. , 2002, Microbes and infection.

[24]  C Caux,et al.  Immunobiology of dendritic cells. , 2000, Annual review of immunology.

[25]  Julia Romanova,et al.  Influenza A mutant viruses with altered NS1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins 1beta and 18. , 2005, The Journal of general virology.

[26]  M. Colonna,et al.  Dendritic cells respond to influenza virus through TLR7‐ and PKR‐independent pathways , 2005, European journal of immunology.

[27]  O. Dittrich‐Breiholz,et al.  Multiple control of interleukin‐8 gene expression , 2002, Journal of leukocyte biology.

[28]  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.

[29]  Quynh-Mai Pham,et al.  Dendritic cells maximize the memory CD8 T cell response to infection. , 2005, Immunity.

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

[31]  Ye Zheng,et al.  Dendritic cell development and survival require distinct NF-kappaB subunits. , 2002, Immunity.

[32]  Ryuji Fukuda,et al.  Mutant Influenza Viruses with a Defective NS1 Protein Cannot Block the Activation of PKR in Infected Cells , 1999, Journal of Virology.

[33]  Ye Zheng,et al.  Dendritic Cell Development and Survival Require Distinct NF-κB Subunits , 2002 .

[34]  R. Steinman,et al.  Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. , 1994, Immunology.

[35]  S. Sealfon,et al.  Accuracy and calibration of commercial oligonucleotide and custom cDNA microarrays. , 2002, Nucleic acids research.

[36]  A. García-Sastre,et al.  Influenza vaccines: present and future. , 2002, The Journal of clinical investigation.

[37]  Antonio Lanzavecchia,et al.  Distinct patterns and kinetics of chemokine production regulate dendritic cell function , 1999, European journal of immunology.

[38]  F. Sallusto,et al.  Origin, maturation and antigen presenting function of dendritic cells. , 1997, Current opinion in immunology.

[39]  A. García-Sastre,et al.  Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins. , 2005, The Journal of general virology.

[40]  Persephone Borrow,et al.  Viral infection switches non-plasmacytoid dendritic cells into high interferon producers , 2003, Nature.

[41]  B. Williams,et al.  Type I interferon induction pathway, but not released interferon, participates in the maturation of dendritic cells induced by negative-strand RNA viruses. , 2003, The Journal of infectious diseases.

[42]  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.

[43]  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.

[44]  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.

[45]  Ira Mellman,et al.  Dendritic Cells Specialized and Regulated Antigen Processing Machines , 2001, Cell.

[46]  F. Lemonnier,et al.  Adjuvant Activities of Novel Cytokines, Interleukin-23 (IL-23) and IL-27, for Induction of Hepatitis C Virus-Specific Cytotoxic T Lymphocytes in HLA-A*0201 Transgenic Mice , 2004, Journal of Virology.

[47]  S. Finke,et al.  Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus , 2005, Journal of Virology.

[48]  S. Akira,et al.  Selective contribution of IFN-alpha/beta signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. , 2003, Proceedings of the National Academy of Sciences of the United States of America.

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

[50]  M. Graham,et al.  Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection , 1994, The Journal of experimental medicine.

[51]  R. Steinman,et al.  The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells In Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation , 2004, The Journal of experimental medicine.