SH3 Binding Motif 1 in Influenza A Virus NS1 Protein Is Essential for PI3K/Akt Signaling Pathway Activation

ABSTRACT Recent studies have demonstrated that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by binding of influenza NS1 protein to the p85 regulatory subunit of PI3K. Our previous study proposed that two polyproline motifs in NS1 (amino acids 164 to 167 [PXXP], SH3 binding motif 1, and amino acids 213 to 216 [PPXXP], SH3 binding motif 2) may mediate binding to the p85 subunit of PI3K. Here we performed individual mutational analyses on these two motifs and demonstrated that SH3 binding motif 1 contributes to the interactions of NS1 with p85β, whereas SH3 binding motif 2 is not required for this process. Mutant viruses carrying NS1 with mutations in SH3 binding motif 1 failed to interact with p85β and induce the subsequent activation of PI3K/Akt pathway. Mutant virus bearing mutations in SH3 binding motif 2 exhibited similar phenotype as the wild-type (WT) virus. Furthermore, viruses with mutations in SH3 binding motif 1 induced more severe apoptosis than did the WT virus. Our data suggest that SH3 binding motif 1 in NS1 protein is required for NS1-p85β interaction and PI3K/Akt activation. Activation of PI3K/Akt pathway is beneficial for virus replication by inhibiting virus induced apoptosis through phosphorylation of caspase-9.

[1]  R. Krug,et al.  The influenza virus NS1 protein: a novel inhibitor of pre-mRNA splicing. , 1994, Genes & development.

[2]  R. Krug,et al.  A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. , 2007, Virology.

[3]  J. Klein,et al.  Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling. , 2003, The Journal of biological chemistry.

[4]  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.

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

[6]  G. Cooper,et al.  Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. , 1995, Science.

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

[8]  R. Krug,et al.  Two functional domains of the influenza virus NS1 protein are required for regulation of nuclear export of mRNA , 1994, Journal of virology.

[9]  F. Alt,et al.  Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85alpha. , 1999, Science.

[10]  S. R. Datta,et al.  Cellular survival: a play in three Akts. , 1999, Genes & development.

[11]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[12]  J. Klein,et al.  Positive and Negative Roles of p85α and p85β Regulatory Subunits of Phosphoinositide 3-Kinase in Insulin Signaling* , 2003, Journal of Biological Chemistry.

[13]  S. Volinia,et al.  Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase. , 1995, Science.

[14]  J. Mackenzie,et al.  The influenza viruses , 2006, The Medical journal of Australia.

[15]  Y. Matsuzawa,et al.  Increased insulin sensitivity and hypoglycaemia in mice lacking the p85α subunit of phosphoinositide 3–kinase , 1999, Nature Genetics.

[16]  R. Krug,et al.  The 3'-end-processing factor CPSF is required for the splicing of single-intron pre-mRNAs in vivo. , 2001, RNA.

[17]  Thorsten Wolff,et al.  Bivalent role of the phosphatidylinositol‐3‐kinase (PI3K) during influenza virus infection and host cell defence , 2006, Cellular microbiology.

[18]  Andrew Macdonald,et al.  The Hepatitis C Virus NS5A Protein Activates a Phosphoinositide 3-Kinase-dependent Survival Signaling Cascade* , 2004, Journal of Biological Chemistry.

[19]  N. Hay,et al.  The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. , 1997, Genes & development.

[20]  Johannes G. Bode,et al.  Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses , 2007, Journal of Virology.

[21]  R. Lamb,et al.  Segment 8 of the influenza virus genome is unique in coding for two polypeptides. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

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

[23]  R. Krug,et al.  The influenza virus NS1 protein binds to a specific region in human U6 snRNA and inhibits U6-U2 and U6-U4 snRNA interactions during splicing. , 1995, RNA.

[24]  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.

[25]  L. Babiuk,et al.  Up-regulation of fatty acid synthase promoter by hepatitis C virus core protein: genotype-3a core has a stronger effect than genotype-1b core. , 2007, Journal of hepatology.

[26]  R. Webster,et al.  Eight-plasmid system for rapid generation of influenza virus vaccines. , 2002, Vaccine.

[27]  Yan Zhou,et al.  Influenza A virus NS1 protein activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by direct interaction with the p85 subunit of PI3K. , 2007, The Journal of general virology.

[28]  B. G. Hale,et al.  PI3K signalling during influenza A virus infections. , 2007, Biochemical Society transactions.

[29]  Yan Zhou,et al.  Effect of the phosphatidylinositol 3-kinase/Akt pathway on influenza A virus propagation. , 2007, The Journal of general virology.

[30]  S. Volinia,et al.  Phosphatidylinositol 3-kinase: Structure and expression of the 110 kd catalytic subunit , 1992, Cell.

[31]  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.

[32]  David R. Kaplan,et al.  Regulation of Neuronal Survival by the Serine-Threonine Protein Kinase Akt , 1997, Science.

[33]  L. Cantley,et al.  Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit. , 1993, The Journal of biological chemistry.

[34]  John Calvin Reed,et al.  Regulation of cell death protease caspase-9 by phosphorylation. , 1998, Science.

[35]  Richard E. Randall,et al.  Influenza A virus NS1 protein binds p85β and activates phosphatidylinositol-3-kinase signaling , 2006, Proceedings of the National Academy of Sciences.

[36]  A. Toker,et al.  Signalling through the lipid products of phosphoinositide-3-OH kinase , 1997, Nature.

[37]  T. Pawson,et al.  SH2 domains recognize specific phosphopeptide sequences , 1993, Cell.

[38]  A. Ullrich,et al.  Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases , 1991, Cell.

[39]  Michael Gale,et al.  Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A Virus , 2006, Journal of Virology.

[40]  R. Webster,et al.  A DNA transfection system for generation of influenza A virus from eight plasmids. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  R. Lamb.  Genes and Proteins of the Influenza Viruses , 1989 .

[42]  R. Chagpar,et al.  A-Raf associates with and regulates platelet-derived growth factor receptor signalling. , 2005, Cellular signalling.

[43]  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.