, Volume 12, Issue 8, pp 1419–1432 | Cite as

Control of apoptosis in influenza virus-infected cells by up-regulation of Akt and p53 signaling

  • Oleg P. ZhirnovEmail author
  • Hans-Dieter Klenk
Original Paper


PI3k-Akt and p53 pathways are known to play anti- and pro-apoptotic roles in cell death, respectively. Whether these pathways are recruited in influenza virus infection in highly productive monkey (CV-1) and canine (MDCK) kidney cells was studied here. Phosphorylation of Akt (Akt-pho) was found to occur only early after infection (5–9 h.p.i). Nuclear accumulation and phosphorylation of p53 (p53-pho), and expression of its natural target p21/waf showed low constitutive levels at this period, whereas all three parameters were markedly elevated at the late apoptotic stage (17–20 h.p.i.). Up-regulation of Akt-pho and p53-pho was not induced by UV-inactivated virus suggesting that it required virus replication. Also, mRNAs of p53 and its natural antagonist mdm2 were not increased throughout infection indicating that p53-pho was up-regulated by posttranslational mechanisms. However, p53 activation did not seem to play a leading role in influenza-induced cell death: (i) infection of CV1 and MDCK cells with recombinant NS1-deficient virus provoked accelerated apoptotic death characterized by the lack of p53 activation; (ii) mixed apoptosis-necrosis death developed in influenza-infected human bronchial H1299 cells carrying a tetracycline-regulated p53 gene did not depend on p53 gene activation by tetracycline. Virus-induced apoptosis and signaling of Akt and p53 developed in IFN-deficient VERO cells with similar kinetics as in IFN-competent CV1-infected cells indicating that these processes were endocrine IFN-independent. Apoptosis in influenza-infected CV-1 and MDCK cells was Akt-dependent and was accelerated by Ly294002, a specific inhibitor of PI3k-Akt signaling, and down-regulated by the viral protein NS1, an inducer of host Akt. The obtained data suggest that influenza virus (i) initiates anti-apoptotic PI3k-Akt signaling at early and middle phases of infection to protect cells from fast apoptotic death and (ii) provokes both p53-dependent and alternative p53-independent apoptotic and/or necrotic (in some host systems) cell death at the late stage of infection.


Influenza virus Apoptosis Akt p53 



The authors thank Dr. Peter Chumakov from the Engelhard Institute of Molecular Biology (Moscow) and Prof. Matthias Dobbelstein, Dr. Michael Schuemann, and Dr. Francoise Debierre-Grockiego from the Institute of Virology, Philipps University of Marburg, for useful discussions and help with the experiments. The authors acknowledge Dr. Irina Vorobjeva for assistance with the purification of DNA plasmids. This work was supported by SFB 593 program and Grant 04-48290 of Russian Foundation for Basic research.


  1. 1.
    Fesq H, Bacher M, Nain M, Gemsa D (1994) Programmed cell death (apoptosis) in human monocytes infected by influenza A virus. Immunobiology 190:175–182PubMedGoogle Scholar
  2. 2.
    Hinshaw VG, Olsen CW, Dybdahi-Sissoko N, Evans D (1994) Apoptosis: a mechanism of cell killing by influenza A and B viruses. J Virol 68:3667–3673PubMedGoogle Scholar
  3. 3.
    Takizawa T, Matsukawa S, Higuchi Y, Nakamura S, Nakanishi Y, Fukuda R (1993) Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells. J Gen Virol 74:2347–2355PubMedGoogle Scholar
  4. 4.
    Arndt U, Wennemuth G, Barth P, Nain M, Al-Abed Y, Meinhardt A, Gemsa D, Bacher M (2002) Release of macrophage migration inhibitory factor and CXCL8/interleukin-8 from lung epithelial cells rendered necrotic by influenza A virus infection. J Virol 76:9298–9306PubMedCrossRefGoogle Scholar
  5. 5.
    Seo SH, Webby R, Webster RG (2004) No apoptotic deaths and different levels of inductions of inflammatory cytokines in alveolar macrophages infected with influenza viruses. Virology 329:270–279PubMedGoogle Scholar
  6. 6.
    Zhirnov OP, Klenk HD (2003) Human influenza viruses are proteolytically activated and do not induce apoptosis in CACO-2 cells. Virology 313:198–212PubMedCrossRefGoogle Scholar
  7. 7.
    Balachandran S, Roberts PC, Kipperman T, Bhalla KN, Compans RW, Archer DR, Barber GN (2000) Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/caspase-8 death signaling pathway. J Virol 74:1513–1523PubMedCrossRefGoogle Scholar
  8. 8.
    Takizawa T, Tatematsu C, Ohashi K, Nakanishi Y (1999) Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death. Microbiol Immunol 43:245–252PubMedGoogle Scholar
  9. 9.
    Zhirnov OP, Konakova TE, Wolff T, Klenk HD (2002) NS1 protein of influenza A virus down-regulates apoptosis. J Virol 76:1617–1625PubMedCrossRefGoogle Scholar
  10. 10.
    Stray SJ, Air GM (2001) Apoptosis by influenza viruses correlates with efficiency of viral mRNA synthesis. Virus Res 77:3–17PubMedCrossRefGoogle Scholar
  11. 11.
    Wurzer WJ, Planz O, Ehrhardt C, Giner M, Silberzohn T, Pleschka S, Ludwig S (2003) Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J 22:2717–2718PubMedCrossRefGoogle Scholar
  12. 12.
    Wurzer WJ, Ehrhardt C, Pleschka S, Berberich-Siebelt F, Wolff T, Walczak H, Planz O, Ludwig S (2004) NF-kB dependent induction of TRAIL and Fas/FasL is crucial for efficient influenza virus propagation. J Biol Chem 279:30931–30937PubMedCrossRefGoogle Scholar
  13. 13.
    Barber GN (2001) Host defense, viruses and apoptosis. Cell Death Dif 8:113–126CrossRefGoogle Scholar
  14. 14.
    Kurokawa M, Koyama AH, Yasuoka S, Adachi A (1999) Influenza virus overcomes apoptosis by rapid multiplication. Int J Mol Med 3:527–530PubMedGoogle Scholar
  15. 15.
    Balachandran S, Kim CN, Yeh WC, Mak TW, Bhalla K, Barber GN (1998) Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signalling. EMBO J 17:6888–6902PubMedCrossRefGoogle Scholar
  16. 16.
    Gern JE, French DA, Grindle KA, Brockman-Schneider RA, Konno S, Busse WW (2003) Double-stranded RNAinduces the synthesis of specific chemokines by bronchial epithelial cells. Amer J Resp Cell Mol Biol 28:731–737CrossRefGoogle Scholar
  17. 17.
    Takizawa T, Ohashi K, Nakanishi Y (1996) Possible involvement of dsRNA-activated protein kinase in cell death by influenza virus infection. J Virol 70:8128–8132PubMedGoogle Scholar
  18. 18.
    Uetani K, Der SD, Zamanian-Daryoush M, de La Motte C, Lieberman BY, Williams BR, Erzurum SC (2000) Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase. J Immunol 165:988–996PubMedGoogle Scholar
  19. 19.
    Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii KJ, Yamaguchi O, Otsu K, Tsujimura T, Koh CS, Reise Sousa C, Matsuura Y, Fujita T, Akira S (2006) Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441(7089):101–105PubMedCrossRefGoogle Scholar
  20. 20.
    Guo Z, Chen LM, Zeng H, Gomez JA, Plowden J, Fujita T, Katz JM, Donis RO, Sambhara S (2006) NS1 Protein of Influenza A Virus Inhibits the Function of Intracytoplasmic Pathogen Sensor, RIG-I. Am J Respir Cell Mol Biol Oct 19; [Epub ahead of print]Google Scholar
  21. 21.
    Hiscott J, Kwon H, Génin P (2001) Hostile takeovers: viral appropriation of the NF-κB pathway. Jclin Invest 107:143–151Google Scholar
  22. 22.
    Morris SJ, Price GE, Barnett JM, Hiscox SA, Smith H, Sweet C (1999) Role of neuraminidase in influenza virus-induced apoptosis. J Gen Virol 80:137–146PubMedGoogle Scholar
  23. 23.
    Schultz-Cherry S, Hinshaw VS (1996) Influenza virus neuraminidase activates latent transforming growth factor beta. J Virol 70:8624–8629PubMedGoogle Scholar
  24. 24.
    Chanturiya AN, Basanez G, Schubert U, Henklein P, Yewdell JW, Zimmerberg J (2004) PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein, creates variably sized pores in planar lipid membranes. J Virol 78:6304–6312PubMedCrossRefGoogle Scholar
  25. 25.
    Chen WS, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, Basta S, OTNeill R, Schickli J, Palese P, Henklein P, Bennink JR, Yewdell JW (2001) A novel influenza A virus mitochondrial protein that induces cell death. Nat Med 7:1306–1312PubMedCrossRefGoogle Scholar
  26. 26.
    Gibbs JS, Malide D, Hornung F, Bennink JR, Yewdell JW (2003) The influenza A virus PB1-F2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function. J Virol 77:7214–7224PubMedCrossRefGoogle Scholar
  27. 27.
    Timofeeva TA, Klenk HD, Zhirnov OP (2001) Identification of the protease-binding domain in the N-terminal region of influenza virus A matrix protein M1. Mol Biol (Russian) 35:411–416CrossRefGoogle Scholar
  28. 28.
    Zhirnov OP, Ksenofontov AL, Kuzmina SG, Klenk HD (2002). Interaction of influenza A virus M1 matrix protein with caspases. Biochemistry (Russian) 67:534–539Google Scholar
  29. 29.
    Morris SJ, Smith H, Sweet C (2002) Exploitation of the Herpes simplex virus translocating protein VP22 to carry influenza virus proteins into cells for studies of apoptosis: direct confirmation that neuraminidase induces apoptosis and indications that other proteins may have a role. Arch Virol 147:961–979PubMedCrossRefGoogle Scholar
  30. 30.
    Ludwig S, Wang X, Ehrhardt C, Zheng H, Donelan N, Planz O, Pleschka S, Garcia-Sastre A, Heins G, Wolff T (2002) The influenza A virus NS1 protein inhibits activation of the jun N-terminal kinase and AP-1 transcription factors. J Virol 76:11166–11171PubMedCrossRefGoogle Scholar
  31. 31.
    Fujimoto I, Takizawa T, Ohba Y, Nakanishi Y (1998) Co-expression of Fas and Fas-ligand on the surface of influenza virus-infected cells. Cell Death Differ 5:426–431PubMedCrossRefGoogle Scholar
  32. 32.
    Wada N, Matsumura M, Oba Y, Kobayashi N, Takizawa T, Nakanishi Y (1995) Transcription stimulation of the Fas-encoding gene by nuclear factor for interleukin-6 expression upon influenza virus infection. J Biol Chem 270:18007–18012PubMedCrossRefGoogle Scholar
  33. 33.
    Olsen CW, Kehren JC, Dybdahl-Sissoko NR, Hinshaw VS (1996) bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation. J Virol 70(1):663–666PubMedGoogle Scholar
  34. 34.
    Oh S, McCaffery JM, Eichelberger MC (2000) Dose-dependent changes in influenza virus-infected dendritic cells result in increased allogeneic T-cell proliferation at low, but not high, doses of virus. J Virol 74(12):5460–5469PubMedCrossRefGoogle Scholar
  35. 35.
    Lin C, Zimmer SG, Lu Z, Holland RE Jr, Dong Q, Chambers TM (2001) The involvement of a stress-activated pathway in equine influenza virus-mediated apoptosis. Virology 287:202–213PubMedCrossRefGoogle Scholar
  36. 36.
    Bernasconi D, Amici C, La Frazia S, Ianaro A, Santoro MG (2005) The IkappaB kinase is a key factor in triggering influenza A virus-induced inflammatory cytokine production in airway epithelial cells. J Biol Chem 280:24127–24134PubMedCrossRefGoogle Scholar
  37. 37.
    Maruoka S, Hashimoto S, Gon Y, Nishitoh H, Takeshita I, Asai Y, Mizumura K, Shimizu K, Ichijo H, Horie T 2003. ASK1 regulates influenza virus infection-induced apoptotic cell death. Biochem Biophys Res Commun 307(4):870–876PubMedCrossRefGoogle Scholar
  38. 38.
    Takizawa T, Tatematsu C, Nakanishi Y (2002) Double-stranded RNA-activated protein kinase interacts with apoptosis signal-regulating kinase 1. Implications for apoptosis signaling pathways. Eur J Biochem 269(24):6126–6132PubMedCrossRefGoogle Scholar
  39. 39.
    Edelmann KH, Richardson-Burns S, Alexopoulou L, Tyler KL, Flavell RA, Oldstone MBA (2004) Does Toll-like receptor 3 play a biological role in virus infections? Virology 322:231–238PubMedCrossRefGoogle Scholar
  40. 40.
    Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S (2004) Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526–1529PubMedCrossRefGoogle Scholar
  41. 41.
    Lund JM, Alexpoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA (2004) Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 101:5598–5603PubMedCrossRefGoogle Scholar
  42. 42.
    Technau-Ihling K, Ihling C, Kromeir J, Brandner J (2001) Influenza A virus infection of mice induces nuclear accumulation of the tumor-suppressor protein p53 in the lung. Arch Virol 146:1655–1666PubMedCrossRefGoogle Scholar
  43. 43.
    Turpin E, Luke K, Jones J, Tumpey T, Konan K, Schultz-Cherry S (2005) Influenza virus infection increases p53 activity: role of p53 in cell death and viral replication. J Virol 79:8802–8811PubMedCrossRefGoogle Scholar
  44. 44.
    Brydon EWA, Smith H, Sweet C (2003) Influenza A virus-induced apoptosis in bronchiolar epithelial (NCI-H292) cells limits proinflammatory cytokine release. J Gen Virol 84:2389–2400PubMedCrossRefGoogle Scholar
  45. 45.
    Tyner JW, Uchida O, Kajiwara N, Kim EY, Patel AC, O’Sullivan MP, Walter MJ, Schwendener RA, Cook DN, Danoff TM, Holtzman MJ (2005) CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection. Nat Med 11:1180–1187PubMedCrossRefGoogle Scholar
  46. 46.
    McKinney LC, Galliger SJ, Lowy RJ (2003) Active and inactive influenza virus induction of tumor necrosis factor-alpha and nitric oxide in J774.1 murine macrophages: modulation by interferon-gamma and failure to induce apoptosis. Virus Res 97:117–126PubMedCrossRefGoogle Scholar
  47. 47.
    Pleschka S, Wolff T, Ehrhardt C, Hobom G, Planz O, Rapp UR, Ludwig S (2001) Influenza virus propagation is impaired by inhibition of the Raf/MEK/ERK signalling cascade. Nat Cell Biol 3(3):301–305PubMedCrossRefGoogle Scholar
  48. 48.
    Nimmerjahn F, Dudziak D, Dirmeier U, Hobom G, Riedel A, Schlee M, Staudt LM, Rosenwald A, Behrends U, Bornkamm GW, Mautner J (2004) Active NF-kappaB signalling is a prerequisite for influenza virus infection. J Gen Virol 85:2347–2356PubMedCrossRefGoogle Scholar
  49. 49.
    Garcia-Sastre A, Egorov A, Matassov D, Brandt S, Levy ED, Durbin JE, Palese P, Muster T (1998) Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252:324–330PubMedCrossRefGoogle Scholar
  50. 50.
    Chen X, Ko LJ, Jayaraman L, Prives C (1996) p53 levels, functional domains, and DNA damage determine the extent of the apoptotic response of tumor cells. Genes Dev 10:2438PubMedCrossRefGoogle Scholar
  51. 51.
    Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108(2):193–199PubMedCrossRefGoogle Scholar
  52. 52.
    Zhirnov OP, Konakova TE, Wgarten W, Klenk H-D (1999) Caspase-dependent N-terminal cleavage of influenza virus nucleocapsid protein in infected cells. J Virol 73:10158–10163PubMedGoogle Scholar
  53. 53.
    Contente A, Dittmer A, Koch MC, Roth J, Dobbelstein M (2002) A polymorphic microsatellite that mediates induction of PIG3 by p53. Nat Genet 30:315–320PubMedCrossRefGoogle Scholar
  54. 54.
    McGahon AJ, Martin SJ, Bissonnette RP, Mahboubi A, Shi Y, Mogil RJ, Nishioka WK, Green DR (1995) The end of the (cell) line - methods for the study of apoptosis in vitro. Methods Cell Biol 46:153–185PubMedCrossRefGoogle Scholar
  55. 55.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  56. 56.
    Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, Sasaki S, Imai K, Shibue T, Honda K, Taniguchi T (2003) Integration of interferon-alpha/beta signaling to p53 responses in tumour suppression and antiviral defense. Nature 424(6948):516–523PubMedCrossRefGoogle Scholar
  57. 57.
    Diaz MO, Zeimin S, Le Beau MM, Pitha P, Smith SD, Chilcote RR, Rowley JD (1988) Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines. Proc Natl Acad Sci USA 85:5259–5263PubMedCrossRefGoogle Scholar
  58. 58.
    Bode AM, Dong Z (2004) Post-translational modification of p53 in tumorigenesis Nat Rev Cancer 4(10):793–805PubMedCrossRefGoogle Scholar
  59. 59.
    Yang XJ (2005) Multisite protein modification and intramolecular signaling. Oncogene 24(10):1653–1662PubMedCrossRefGoogle Scholar
  60. 60.
    Bond GL, Hu W, Levine AJ (2005) MDM2 is a central node in the p53 pathway: 12 years and counting. Curr Cancer Drug Targets 5:3–8PubMedCrossRefGoogle Scholar
  61. 61.
    Downward J (2004) PI-3 kinase, Akt and cell survival. Cell Dev Biol 15: 177–182CrossRefGoogle Scholar
  62. 62.
    Davies SP, Reddy H, Caivano M, Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351(Pt 1):95–105PubMedCrossRefGoogle Scholar
  63. 63.
    Brydon EW, Morris SJ, Sweet C (2005) Role of apoptosis and cytokines in influenza virus morbidity. FEMS Microbiol Rev 29:837–50PubMedCrossRefGoogle Scholar
  64. 64.
    Yee KS, Vousden KH (2005) Complicating the complexity of p53. Carcinogenesis 26:1317–22PubMedCrossRefGoogle Scholar
  65. 65.
    Muster T, Rajtarova J, Sachet M, Unger H, Fleischhacker R, Romirer I, Grassauer A, Url A, Garcia-Sastre A, Wolff K, Pehamberger H, Bergmann M (2004) Interferon resistance promotes oncolysis by influenza virus NS1-deletion mutants. Int J Cancer 110(1):15–21PubMedCrossRefGoogle Scholar
  66. 66.
    Inglis SC, Carroll AR, Lamb RA, Mahy BWJ (1976) Polypeptides specified by the influenza virus genome. 1. Evidence for eight distinct gene products specified by fowl plague virus. Virology 74:489–503PubMedCrossRefGoogle Scholar
  67. 67.
    Skehel JJ (1973) Early polypeptide synthesis in influenza virus-infected cells. Virology 56:394–399PubMedCrossRefGoogle Scholar
  68. 68.
    Ehrhardt C, Marjuki H, Wolff T, Nurnberg B, Planz O, Pleschka S, Ludwig S (2006) Bivalent role of the phosphatidylinositol-3-kinase (PI3K) during influenza virus infection and host cell defence. Cell Microbiol 8(8):1336–48PubMedCrossRefGoogle Scholar
  69. 69.
    Hale BG, Jackson D, Chen YH, Lamb RA, Randall RE (2006) Influenza A virus NS1 protein binds p85beta and activates phosphatidylinositol-3-kinase signaling. Proc Natl Acad Sci USA 103(38):14194–9PubMedCrossRefGoogle Scholar
  70. 70.
    Leers MP, Kolgen W, Bjorklund V, Bergman T, Tribbick G, Persson B, Bjorklund P, Ramaekers FC, Bjorklund B, Nap M, Jornvall H, Schutte B (1999) mmunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 187:567–572 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.D.I. Ivanovsky Institute of VirologyMoscowRussia
  2. 2.Institute of VirologyPhilipps University of MarburgMarburgGermany

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