Dengue virus (DENV) is the causative agent of dengue fever and the more severe forms of the infection known as dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). Secondary infections with a serotype different from the primary infection are considered a risk factor for the development of DHF/DSS. One explanation for the increased risk of DHF/DSS development after heterologous secondary infections is the antibody-dependent enhancement (ADE) hypothesis. This hypothesis postulates that pre-existing non-neutralizing antibodies will form immune complexes with the new serotype-infecting virus that in turn will have enhanced capacity to infect macrophages and other Fc receptor (FcR)-bearing cells. Despite the evidence supporting the ADE hypothesis, the molecular mechanisms of ADE are not fully understood. In this work, we present evidence which indicates that intact lipid rafts are required for the ADE infection of U937 cells with DENV. Flow cytometry analysis to measure the percentage of infected cells showed that treatment of differentiated U937 cells with nystatin (30 μg ml), filipin (10 μg ml) or -methyl cyclodextrin (30 mM) significantly reduces (<0.05) the ADE of DENV-4 infection without any effect on viability or the number of FcR-bearing cells. Later cholesterol replenishment by supplementing treated cell cultures with bovine fetal serum for 24 h re-established lipid raft integrity and reversed the alteration of the ADE (<0.05). Our results suggest that ADE of U937 infection by DENV requires the presence of cholesterol and cholesterol-rich membrane microdomains.


Article metrics loading...

Loading full text...

Full text loading...



  1. Acosta, E. G., Castilla, V. & Damonte, E. B.(2009). Alternative infectious entry pathways for dengue virus serotypes into mammalian cells. Cell Microbiol 11, 1533–1549.[CrossRef] [Google Scholar]
  2. Aderem, A. & Underhill, D. M.(1999). Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17, 593–623.[CrossRef] [Google Scholar]
  3. Barabé, F., Rollet-Labelle, E., Gilbert, C., Fernandes, M., Naccache, S. & Naccache, P.(2002). Early Events in the activation of FcγRIIA in human neutrophils: stimulated insolubilization, translocation to detergent-resistant domains, and degradation of FcγRIIA. J Immunol 168, 4042–4049.[CrossRef] [Google Scholar]
  4. Barman, S., Adhikary, L., Chakrabarti, A. K., Bernas, C., Kawaoka, Y. & Nayak, D. P.(2004). Role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza A virus neuraminidase in raft association and virus budding. J Virol 78, 5258–5269.[CrossRef] [Google Scholar]
  5. Blank, N., Schiller, M., Krienke, S., Wabnitz, G., Ho, A. D. & Lorenz, H. M.(2007). Cholera toxin binds to lipid rafts but has a limited specificity for ganglioside GM1. Immunol Cell Biol 85, 378–382.[CrossRef] [Google Scholar]
  6. Bournazos, S., Hart, S. P., Chamberlain, L. H., Glennie, M. J. & Dransfield, I.(2009). Association of FcγRIIa (CD32a) with lipid rafts regulates ligand binding activity. J Immunol 182, 8026–8036.[CrossRef] [Google Scholar]
  7. Brandt, W. E., McCown, J. M., Gentry, M. K. & Russell, P. K.(1982). Infection enhancement of dengue Type 2 virus in the U-937 human monocyte cell line by antibodies to flavivirus cross-reactive determinants. Infect Immun 36, 1036–1041. [Google Scholar]
  8. Cambi, A., de Lange, F., van Maarseveen, N. M., Nijhuis, M., Joosten, B., van Dijk, E. M., de Bakker, B. I., Fransen, J. A., Bovee-Geurts, P. H. & other authors(2004). Microdomains of the C-type lectin DC-SIGN are portals for virus entry into dendritic cells. J Cell Biol 164, 145–155.[CrossRef] [Google Scholar]
  9. Chareonsirisuthigul, T., Kalayanarooj, S. & Ubol, S.(2007). Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells. J Gen Virol 88, 365–375.[CrossRef] [Google Scholar]
  10. Chen, S. T., Lin, Y. L., Huang, M. T., Wu, M. F., Cheng, S. C., Lei, H. Y., Lee, C. K., Chiou, T. W., Wong, C. H. & Hsieh, S. L.(2008). CLEC5A is critical for dengue-virus-induced lethal disease. Nature 453, 672–676.[CrossRef] [Google Scholar]
  11. Chu, J. J. & Ng, M. L.(2004). Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J Virol 78, 10543–10555.[CrossRef] [Google Scholar]
  12. Chu, C. L., Buczek-Thomas, J. A. & Nugent, M. A.(2004). Heparan sulphate proteoglycans modulate fibroblast growth factor-2 binding through a lipid raft-mediated mechanism. Biochem J 379, 331–341.[CrossRef] [Google Scholar]
  13. del Real, G., Jiménez-Baranda, S., Mira, E., Lacalle, R. A., Lucas, P., Gómez-Moutón, C., Alegret, M., Peña, J. M., Rodríguez-Zapata, M. & other authors(2004). Statins inhibit HIV-1 infection by down-regulating Rho activity. J Exp Med 200, 541–547.[CrossRef] [Google Scholar]
  14. Diakonova, M., Bokoch, G. & Swanson, J. A.(2002). Dynamics of cytoskeletal proteins during Fcγ receptor-mediated phagocytosis in macrophages. Mol Biol Cell 13, 402–411.[CrossRef] [Google Scholar]
  15. García-García, E. & Rosales, C.(2002). Signal transduction during Fc receptor-mediated phagocytosis. J Leukoc Biol 72, 1092–1108. [Google Scholar]
  16. García-García, E., Brown, E. & Rosales, C.(2007). Transmembrane mutations to FcγRIIA alter its association with lipid rafts: implications for receptor signaling. J Immunol 178, 3048–3058.[CrossRef] [Google Scholar]
  17. Goncalvez, A. P., Engle, R. E., St Claire, M., Purcell, R. H. & Lai, C.-J.(2007). Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proc Natl Acad Sci U S A 104, 9422–9427.[CrossRef] [Google Scholar]
  18. Gubler, D. J.(2006). Dengue/dengue haemorrhagic fever: history and current status. Novartis Found Symp 277, 3–16. [Google Scholar]
  19. Halstead, S. B., Nimmannitya, S. & Cohen, S. N.(1970). Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J Biol Med 42, 311–328. [Google Scholar]
  20. Hancock, J. F.(2006). Lipid rafts: contentious only from simplistic standpoints. Nat Rev Mol Cell Biol 7, 456–462.[CrossRef] [Google Scholar]
  21. Henchal, E. A., McCown, J. M., Burke, D. S., Seguin, M. C. & Brandt, W. E.(1985). Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies. Am J Trop Med Hyg 34, 162–169. [Google Scholar]
  22. Huang, K.-J., Yang, Y.-C., Lin, Y.-S., Huang, J.-H., Liu, H.-S., Yeh, T.-M., Chen, S.-H., Liu, C.-C. & Lei, H.-Y.(2006). The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol 176, 2825–2832.[CrossRef] [Google Scholar]
  23. Ikeda, M., Abe, K., Yamada, M., Dansako, H., Naka, K. & Kato, N.(2006). Different anti-HCV profiles of statins and their potential for combination therapy with interferon. Hepatology 44, 117–125.[CrossRef] [Google Scholar]
  24. Jin, X.(2008). Cellular and molecular basis of antibody-dependent enhancement in human dengue pathogenesis. Future Virol 3, 343–361.[CrossRef] [Google Scholar]
  25. Kapadia, S. B., Barth, H., Baumert, T., McKeating, J. A. & Chisari, F. V.(2007). Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I. J Virol 81, 374–383.[CrossRef] [Google Scholar]
  26. Katsumata, O., Hara-Yokoyama, M., Sautès-Fridman, C., Nagatsuka, Y., Katada, T., Hirabayashi, Y., Shimizu, K., Fujita-Yoshigaki, J., Sugiya, H. & Furuyama, S.(2001). Association of FcγRII with low-density detergent-resistant membranes is important for cross-linking-dependent initiation of the tyrosine phosphorylation pathway and superoxide generation. J Immunol 167, 5814–5823.[CrossRef] [Google Scholar]
  27. Kliks, S. C., Nimmanitya, S., Nisalak, A. & Burke, D. S.(1988). Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg 38, 411–419. [Google Scholar]
  28. Kliks, S. C., Nisalak, A., Brandt, W. E., Wahl, L. & Burke, D. S.(1989). Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg 40, 444–451. [Google Scholar]
  29. Kono, H., Suzuki, T., Yamamoto, K., Okada, M., Yamamoto, T. & Honda, Z.(2002). Spatial raft coalescence represents an initial step in FcγR signaling. J Immunol 169, 193–203.[CrossRef] [Google Scholar]
  30. Kontny, U., Kurane, I. & Ennis, F. A.(1988). Gamma interferon augments Fcγ receptor-mediated dengue virus infection of human monocytic cells. J Virol 62, 3928–3933. [Google Scholar]
  31. Kou, Z., Quinn, M., Chen, H., Rodrigo, W. W. S. I., Rose, R. C., Schlesinger, J. J. & Jin, X.(2008). Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells. J Med Virol 80, 134–146.[CrossRef] [Google Scholar]
  32. Kurane, I., Kontny, U., Janus, J. & Ennis, F. A.(1990). Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections. Arch Virol 110, 91–101.[CrossRef] [Google Scholar]
  33. Kwiatkowska, K. & Sobota, A.(2001). The clustered Fcγ receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner. Eur J Immunol 31, 989–998.[CrossRef] [Google Scholar]
  34. Kwiatkowska, K., Frey, J. & Sobota, A.(2003). Phosphorylation of FcγRIIA is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts. J Cell Sci 116, 537–550.[CrossRef] [Google Scholar]
  35. Kyle, J. L., Beatty, P. R. & Harris, E.(2007). Dengue virus infects macrophages and dendritic cells in a mouse model of infection. J Infect Dis 195, 1808–1817.[CrossRef] [Google Scholar]
  36. Lee, C. J., Lin, H. R., Liao, C. L. & Lin, Y. L.(2008). Cholesterol effectively blocks entry of flavivirus. J Virol 82, 6470–6480.[CrossRef] [Google Scholar]
  37. Liao, Z., Cimakasky, L. M., Hampton, R., Nguyen, D. H. & Hildreth, J. E.(2001). Lipid rafts and HIV pathogenesis: host membrane cholesterol is required for infection by HIV type 1. AIDS Res Hum Retroviruses 17, 1009–1019.[CrossRef] [Google Scholar]
  38. Lin, Y. W., Wang, K. J., Lei, H. Y., Lin, Y. S., Yeh, T. M., Liu, H. S., Liu, C. C. & Chen, S. H.(2002). Virus replication and cytokine production in dengue virus-infected human B lymphocytes. J Virol 76, 12242–12249.[CrossRef] [Google Scholar]
  39. Lindenbach, B. D. & Rice, C. M.(2003). Molecular biology of flaviviruses. Adv Virus Res 59, 23–61. [Google Scholar]
  40. Littaua, R., Kurane, I. & Ennis, F.(1990). Human IgG Fc receptor II mediates antibody-dependent enhancement of dengue virus infection. J Immunol 144, 3183–3186. [Google Scholar]
  41. Mackenzie, J. M., Khromykh, A. A. & Parton, R. G.(2007). Cholesterol manipulation by West Nile virus perturbs the cellular immune response. Cell Host Microbe 2, 229–239.[CrossRef] [Google Scholar]
  42. Mady, B. J., Erbe, D. V., Kurane, I., Fanger, M. W. & Ennis, F. A.(1991). Antibody-dependent enhancement of dengue virus infection mediated by bispecific antibodies against cell surface molecules other than Fcγ receptors. J Immunol 147, 3139–3144. [Google Scholar]
  43. May, R. C. & Machesky, L. M.(2001). Phagocytosis and the actin cytoskeleton. J Cell Sci 114, 1061–1077. [Google Scholar]
  44. Moi, M. L., Lim, C.-K., Takasaki, T. & Kurane, I.(2010). Involvement of the Fcγ receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection. J Gen Virol 91, 103–111.[CrossRef] [Google Scholar]
  45. Morens, D. M., Halstead, S. B. & Marchette, N. J.(1987). Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb Pathog 3, 231–237.[CrossRef] [Google Scholar]
  46. Mosso, C., Galván-Mendoza, I., Ludert, J. E. & Del Angel, R. M.(2008). Endocytic pathway followed by dengue virus to infect the mosquito cell line C6/36 HT. Virology 378, 193–199.[CrossRef] [Google Scholar]
  47. Philippova, M., Ivanov, D., Joshi, M. B., Kyriakakis, E., Rupp, K., Afonyushkin, T., Bochkov, V., Erne, P. & Resink, T. J.(2008). Identification of proteins associating with glycosylphosphatidylinositol-anchored T-cadherin on the surface of vascular endothelial cells: role for Grp78/BiP in T-cadherin-dependent cell survival. Mol Cell Biol 28, 4004–4017.[CrossRef] [Google Scholar]
  48. Reyes-del Valle, R., Chávez-Salinas, S., Medina, F. & Del Angel, R. M.(2005). Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells. J Virol 79, 4557–4567.[CrossRef] [Google Scholar]
  49. Rodrigo, W. W., Jin, X., Blackley, S. D., Rose, R. C. & Schlesinger, J. J.(2006). Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human FcγRIA (CD64) or FcγRIIA (CD32). J Virol 80, 10128–10138.[CrossRef] [Google Scholar]
  50. Rollet-Labelle, E., Marois, S., Barbeau, K., Malawista, S. E. & Naccache, P. H.(2004). Recruitment of the cross-linked opsonic receptor CD32A (FcγRIIA) to high-density detergent-resistant membrane domains in human neutrophils. Biochem J 381, 919–928.[CrossRef] [Google Scholar]
  51. Rothwell, C., LeBreton, A., Ng, C. Y., Lim, J. Y. H., Liu, W., Vasudevan, S., Labow, M., Gu,, F. & Gaither, L. A.(2009). Cholesterol biosynthesis modulation regulates dengue virus replication. Virology 389, 8–19.[CrossRef] [Google Scholar]
  52. Simons, K. & Ikonen, E.(1997). Functional rafts in cell membranes. Nature 387, 569–572.[CrossRef] [Google Scholar]
  53. Simons, K. & Toomre, D.(2000). Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1, 31–39. [Google Scholar]
  54. Sullivan, N. J.(2001). Antibody-mediated enhancement of viral disease. Curr Top Microbiol Immunol 260, 145–169. [Google Scholar]
  55. Suzuki, T. & Suzuki, Y.(2006). Virus infection and lipid rafts. Biol Pharm Bull 29, 1538–1541.[CrossRef] [Google Scholar]
  56. van Gorp, E. C. M., Suharti, C., Mairuhu, A. T. A., Dolmans, W. M., van der Ven, J., Demacker, P. N. M. & van der Meer, J. W. M.(2002). Changes in the plasma lipid profile as a potential predictor of clinical outcome in dengue hemorrhagic fever. Clin Infect Dis 34, 1150–1153.[CrossRef] [Google Scholar]
  57. Vaughn, D. W., Green, S., Kalayanarooj, S., Innis, B. L., Nimmannitya, S., Suntayakorn, S., Endy, T. P., Raengsakulrach, B., Rothman, A. L. & other authors(2000). Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181, 2–9.[CrossRef] [Google Scholar]
  58. Welsch, S., Miller, S., Romero-Brey, I., Merz, A., Bleck, C. K., Walther, P., Fuller, S. D., Antony, C., Krijnse-Locker, J. & Bartenschlager, R.(2009). Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5, 365–375.[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