Tumor necrosis factor alpha (TNF-) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF- stimulation cannot activate nuclear factor B (NF-B) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-, focussing particularly on cell death and pro-survival signals. TNF- stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF- induced comparable degradation of the inhibitor of NF-B alpha (IB-) and NF-B activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF--induced cell death, TNF--stimulated IB- degradation and NF-B activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF--stimulated survival signals. Transfection experiments demonstrated inhibition of TNF--stimulated NF-B activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF--stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF--stimulated survival pathways and show enhanced susceptibility to TNF--stimulated cell death, suggesting a role for TNF- in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF- to activate NF-B as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF--stimulated NF-B activation and could have important consequences for NF-B-driven release of inflammatory cytokines.


Article metrics loading...

Loading full text...

Full text loading...



  1. Alvarez, S. E., Harikumar, K. B., Hait, N. C., Allegood, J., Strub, G. M., Kim, E. Y., Maceyka, M., Jiang, H., Luo, C. & other authors(2010). Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 465, 1084–1088.[CrossRef] [Google Scholar]
  2. Atrasheuskaya, A., Petzelbauer, P., Fredeking, T. M. & Ignatyev, G.(2003). Anti-TNF antibody treatment reduces mortality in experimental dengue virus infection. FEMS Immunol Med Microbiol 35, 33–42. [CrossRef] [Google Scholar]
  3. Bulich, R. & Aaskov, J. G.(1992). Nuclear localization of dengue 2 virus core protein detected with monoclonal antibodies. J Gen Virol 73, 2999–3003.[CrossRef] [Google Scholar]
  4. Carr, J. M., Hocking, H., Bunting, K., Wright, P. J., Davidson, A., Gamble, J., Burrell, C. J. & Li, P.(2003). Supernatants from dengue virus type-2 infected macrophages induce permeability changes in endothelial cell monolayers. J Med Virol 69, 521–528. [CrossRef] [Google Scholar]
  5. Catteau, A., Kalinina, O., Wagner, M. C., Deubel, V., Courageot, M. P. & Desprès, P.(2003). Dengue virus M protein contains a proapoptotic sequence referred to as ApoptoM. J Gen Virol 84, 2781–2793. [CrossRef] [Google Scholar]
  6. Chen, G. & Goeddel, D. V.(2002). TNF-R1 signaling: a beautiful pathway. Science 296, 1634–1635.[CrossRef] [Google Scholar]
  7. Chen, Y. C. & Wang, S. Y.(2002). Activation of terminally differentiated human monocytes/macrophages by dengue virus: productive infection, hierarchical production of innate cytokines and chemokines, and the synergistic effect of lipopolysaccharide. J Virol 76, 9877–9887.[CrossRef] [Google Scholar]
  8. Chen, H. C., Hofman, F. M., Kung, J. T., Lin, Y. D. & Wu-Hsieh, B. A.(2007). Both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage. J Virol 81, 5518–5526.[CrossRef] [Google Scholar]
  9. Cheng, Y., King, N. J. & Kesson, A. M.(2004). The role of tumor necrosis factor in modulating responses of murine embryo fibroblasts by flavivirus, West Nile. Virology 329, 361–370.[CrossRef] [Google Scholar]
  10. Choi, S. H., Park, K. J., Ahn, B. Y., Jung, G., Lai, M. M. & Hwang, S. B.(2006). Hepatitis C virus nonstructural 5B protein regulates tumor necrosis factor alpha signaling through effects on cellular IκB kinase. Mol Cell Biol 26, 3048–3059.[CrossRef] [Google Scholar]
  11. Clyde, K., Kyle, J. L. & Harris, E.(2006). Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J Virol 80, 11418–11431.[CrossRef] [Google Scholar]
  12. Espina, L. M., Valero, N. J., Hernández, J. M. & Mosquera, J. A.(2003). Increased apoptosis and expression of tumor necrosis factor-alpha caused by infection of cultured human monocytes with dengue virus. Am J Trop Med Hyg 68, 48–53. [Google Scholar]
  13. Gilles, P. N., Fey, G. & Chisari, F. V.(1992). Tumor necrosis factor alpha negatively regulates hepatitis B virus gene expression in transgenic mice. J Virol 66, 3955–3960. [Google Scholar]
  14. Green, S. & Rothman, A.(2006). Immunopathological mechanisms in dengue and dengue hemorrhagic fever. Curr Opin Infect Dis 19, 429–436. [CrossRef] [Google Scholar]
  15. Gualano, R. C., Pryor, M. J., Cauchi, M. R., Wright, P. J. & Davidson, A. D.(1998). Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length cDNA. J Gen Virol 79, 437–446. [Google Scholar]
  16. Halstead, S. B.(2007). Dengue. Lancet 370, 1644–1652. [CrossRef] [Google Scholar]
  17. Herbein, G. & O'Brien, W. A.(2000). Tumor necrosis factor (TNF)-alpha and TNF receptors in viral pathogenesis. Proc Soc Exp Biol Med 223, 241–257.[CrossRef] [Google Scholar]
  18. Hiscott, J., Kwon, H. & Génin, P.(2001). Hostile takeovers: viral appropriation of the NF-κB pathway. J Clin Invest 107, 143–151. [CrossRef] [Google Scholar]
  19. Hober, D., Poli, L., Roblin, B., Gestas, P., Chungue, E., Granic, G., Imbert, P., Pecarere, J. L., Vergez-Pascal, R. & other authors(1993). Serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta) in dengue-infected patients. Am J Trop Med Hyg 48, 324–331. [Google Scholar]
  20. Kittigul, L., Temprom, W., Sujirarat, D. & Kittigul, C.(2000). Determination of tumor necrosis factor-alpha levels in dengue virus infected patients by sensitive biotin-streptavidin enzyme-linked immunosorbent assay. J Virol Methods 90, 51–57. [CrossRef] [Google Scholar]
  21. Koopman, G., Reutelingsperger, C. P., Kuijten, G. A., Keehnen, R. M., Pals, S. T. & van Oers, M. H.(1994). Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415–1420. [Google Scholar]
  22. Kurane, I., Innis, B. L., Nimmannitya, S., Nisalak, A., Meager, A., Janus, J. & Ennis, F. A.(1991). Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue. J Clin Invest 88, 1473–1480. [CrossRef] [Google Scholar]
  23. Li, H. & Lin, X.(2008). Positive and negative signaling components involved in TNFα-induced NF-κB activation. Cytokine 41, 1–8. [CrossRef] [Google Scholar]
  24. Limjindaporn, T., Netsawang, J., Noisakran, S., Thiemmeca, S., Wongwiwat, W., Sudsaward, S., Avirutnan, P., Puttikhunt, C., Kasinrerk, W. & Sriburi, R.(2007). Sensitization to Fas-mediated apoptosis by dengue virus capsid protein. Biochem Biophys Res Commun 362, 334–339.[CrossRef] [Google Scholar]
  25. Luplerdlop, N., Missé, D., Bray, D., Deleuze, V., Gonzalez, J. P., Leardkamolkarn, V., Yssel, H. & Veas, F.(2006). Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction. EMBO Rep 7, 1176–1181. [CrossRef] [Google Scholar]
  26. Marianneau, P., Cardona, A., Edelman, L., Deubel, V. & Desprès, P.(1997). Dengue virus replication in human hepatoma cells activates NF-κB which in turn induces apoptotic cell death. J Virol 71, 3244–3249. [Google Scholar]
  27. Martina, B. E., Koraka, P. & Osterhaus, A. D.(2009). Dengue virus pathogenesis: an integrated view. Clin Microbiol Rev 22, 564–581. [CrossRef] [Google Scholar]
  28. Marusawa, H., Hijikata, M., Chiba, T. & Shimotohno, K.(1999). Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha-mediated apoptosis via NF-κB activation. J Virol 73, 4713–4720. [Google Scholar]
  29. Medin, C. L., Fitzgerald, K. A. & Rothman, A. L.(2005). Dengue virus nonstructural protein NS5 induces interleukin-8 transcription and secretion. J Virol 79, 11053–11061. [CrossRef] [Google Scholar]
  30. Park, K. J., Choi, S. H., Lee, S. Y., Hwang, S. B. & Lai, M. M.(2002). Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation. J Biol Chem 277, 13122–13128. [CrossRef] [Google Scholar]
  31. Pavić, I., Polić, B., Crnković, I., Lucin, P., Jonjić, S. & Koszinowski, U. H.(1993). Participation of endogenous tumour necrosis factor alpha in host resistance to cytomegalovirus infection. J Gen Virol 74, 2215–2223. [CrossRef] [Google Scholar]
  32. Perez, A. B., Sierra, B., Garcia, G., Aguirre, E., Babel, N., Alvarez, M., Sanchez, L., Valdes, L., Volk, H. D. & Guzman, M. G.(2010). Tumor necrosis factor-alpha, transforming growth factor-β1, and interleukin-10 gene polymorphisms: implication in protection or susceptibility to dengue hemorrhagic fever. Hum Immunol 71, 1135–1140. [CrossRef] [Google Scholar]
  33. Pryor, M. J., Carr, J. M., Hocking, H., Davidson, A. D., Li, P. & Wright, P. J.(2001). Replication of dengue virus type 2 in human monocyte-derived macrophages: comparisons of isolates and recombinant viruses with substitutions at amino acid 390 in the envelope glycoprotein. Am J Trop Med Hyg 65, 427–434. [Google Scholar]
  34. Pryor, M. J., Rawlinson, S. M., Butcher, R. E., Barton, C. L., Waterhouse, T. A., Vasudevan, S. G., Bardin, P. G., Wright, P. J., Jans, D. A. & Davidson, A. D.(2007). Nuclear localization of dengue virus nonstructural protein 5 through its importin α/β-recognized nuclear localization sequences is integral to viral infection. Traffic 8, 795–807. [CrossRef] [Google Scholar]
  35. Rawlinson, S. M., Pryor, M. J., Wright, P. J. & Jans, D. A.(2009). CRM1-mediated nuclear export of dengue virus RNA polymerase NS5 modulates interleukin-8 induction and virus production. J Biol Chem 284, 15589–15597.[CrossRef] [Google Scholar]
  36. Roberts, J. L., Moretti, P. A., Darrow, A. L., Derian, C. K., Vadas, M. A. & Pitson, S. M.(2004). An assay for sphingosine kinase activity using biotinylated sphingosine and streptavidin-coated membranes. Anal Biochem 331, 122–129.[CrossRef] [Google Scholar]
  37. Sacco, R., Tsutsumi, T., Suzuki, R., Otsuka, M., Aizaki, H., Sakamoto, S., Matsuda, M., Seki, N., Matsuura, Y. & other authors(2003). Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of caspase-activated DNase. Virology 317, 24–35. [CrossRef] [Google Scholar]
  38. Saito, K., Meyer, K., Warner, R., Basu, A., Ray, R. B. & Ray, R.(2006). Hepatitis C virus core protein inhibits tumor necrosis factor alpha-mediated apoptosis by a protective effect involving cellular FLICE inhibitory protein. J Virol 80, 4372–4379. [CrossRef] [Google Scholar]
  39. Shafee, N. & AbuBakar, S.(2003). Dengue virus type 2 NS3 protease and NS2B-NS3 protease precursor induce apoptosis. J Gen Virol 84, 2191–2195. [CrossRef] [Google Scholar]
  40. Shresta, S., Sharar, K. L., Prigozhin, D. M., Beatty, P. R. & Harris, E.(2006). Murine model for dengue virus-induced lethal disease with increased vascular permeability. J Virol 80, 10208–10217. [CrossRef] [Google Scholar]
  41. Shrestha, B., Zhang, B., Purtha, W. E., Klein, R. S. & Diamond, M. S.(2008). Tumor necrosis factor alpha protects against lethal West Nile virus infection by promoting trafficking of mononuclear leukocytes into the central nervous system. J Virol 82, 8956–8964. [CrossRef] [Google Scholar]
  42. Shrivastava, A., Manna, S. K., Ray, R. & Aggarwal, B. B.(1998). Ectopic expression of hepatitis C virus core protein differentially regulates nuclear transcription factors. J Virol 72, 9722–9728. [Google Scholar]
  43. Souza, D. G., Fagundes, C. T., Sousa, L. P., Amaral, F. A., Souza, R. S., Souza, A. L., Kroon, E. G., Sachs, D., Cunha, F. Q. & other authors(2009). Essential role of platelet-activating factor receptor in the pathogenesis of Dengue virus infection. Proc Natl Acad Sci U S A 106, 14138–14143. [CrossRef] [Google Scholar]
  44. Srikiatkhachorn, A., Ajariyakhajorn, C., Endy, T. P., Kalayanarooj, S., Libraty, D. H., Green, S., Ennis, F. A. & Rothman, A. L.(2007). Virus-induced decline in soluble vascular endothelial growth receptor 2 is associated with plasma leakage in dengue hemorrhagic Fever. J Virol 81, 1592–1600.[CrossRef] [Google Scholar]
  45. Wati, S., Li, P., Burrell, C. J. & Carr, J. M.(2007). Dengue virus (DV) replication in monocyte-derived macrophages is not affected by tumor necrosis factor alpha (TNF-α), and DV infection induces altered responsiveness to TNF-α stimulation. J Virol 81, 10161–10171. [CrossRef] [Google Scholar]
  46. Xia, P., Wang, L., Gamble, J. R. & Vadas, M. A.(1999). Activation of sphingosine kinase by tumor necrosis factor-α inhibits apoptosis in human endothelial cells. J Biol Chem 274, 34499–34505. [CrossRef] [Google Scholar]
  47. Xia, P., Wang, L., Moretti, P. A., Albanese, N., Chai, F., Pitson, S. M., D'Andrea, R. J., Gamble, J. R. & Vadas, M. A.(2002). Sphingosine kinase interacts with TRAF2 and dissects tumor necrosis factor-alpha signaling. J Biol Chem 277, 7996–8003.[CrossRef] [Google Scholar]
  48. Yamane, D., Zahoor, M. A., Mohamed, Y. M., Azab, W., Kato, K., Tohya, Y. & Akashi, H.(2009). Inhibition of sphingosine kinase by bovine viral diarrhea virus NS3 is crucial for efficient viral replication and cytopathogenesis. J Biol Chem 284, 13648–13659. [CrossRef] [Google Scholar]
  49. Yang, J. S., Ramanathan, M. P., Muthumani, K., Choo, A. Y., Jin, S. H., Yu, Q. C., Hwang, D. S., Choo, D. K., Lee, M. D. & other authors(2002). Induction of inflammation by West Nile virus capsid through the caspase-9 apoptotic pathway. Emerg Infect Dis 8, 1379–1384.[CrossRef] [Google Scholar]
  50. Yang, M. R., Lee, S. R., Oh, W., Lee, E. W., Yeh, J. Y., Nah, J. J., Joo, Y. S., Shin, J., Lee, H. W. & other authors(2008). West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus. Cell Microbiol 10, 165–176. [Google Scholar]
  51. Yen, Y.-T., Chen, H.-C., Lin, Y.-D., Shieh, C.-C. & Wu-Hsieh, B.-A.(2008). Enhancement by tumor necrosis factor alpha of dengue virus-induced endothelial cell production of reactive nitrogen and oxygen species is key to hemorrhage development. J Virol 82, 12312–12324. [CrossRef] [Google Scholar]
  52. Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C. & Lai, M. M.(1998). Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. J Virol 72, 3691–3697. [Google Scholar]
  53. Zhu, N., Ware, C. F. & Lai, M. M.(2001). Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor. Virology 283, 178–187. [CrossRef] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error