1887

Abstract

Dengue virus NS1 is a glycoprotein of 46–50 kDa that is conserved among flaviviruses, associates as a dimer to cell membranes and is secreted as a hexamer to the extracellular milieu. Recent evidence showed that NS1 is secreted efficiently from infected mosquito cells. To explore the secretory route of NS1 in mosquito cells, infected cells were treated with brefeldin A (BFA) and methyl-beta-cyclodextrin (MβCD). The results showed that MβCD, but not BFA, significantly reduced the release of NS1. Moreover, silencing the expression of caveolin-1 (CAV1; a key component of the caveolar system that transports cholesterol inside the cell), but not SAR1 (a GTPase that participates in the classical secretory pathway), also results in a significant reduction of the secretion of NS1. These results indicate that NS1 is released from mosquito cells via an unconventional secretory route that bypasses the Golgi complex, with the participation of CAV1. In agreement with this notion, differences were observed in the glycosylation status between secreted NS1 and E proteins. Classical mechanics and docking simulations suggested highly favoured interactions between the caveolin-binding domain present in NS1 and the scaffolding domain of CAV1. Finally, proximity ligation assays showed direct interaction between NS1 and CAV1 in infected mosquito cells. These findings are in line with the lipoprotein nature of secreted NS1 and provide new insights into the biology of NS1.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000881
2017-07-31
2019-09-18
Loading full text...

Full text loading...

/deliver/fulltext/jgv/98/8/2088.html?itemId=/content/journal/jgv/10.1099/jgv.0.000881&mimeType=html&fmt=ahah

References

  1. Muller DA, Young PR. The flavivirus NS1 protein: molecular and structural biology, immunology, role in pathogenesis and application as a diagnostic biomarker. Antiviral Res 2013;98:192–208 [CrossRef][PubMed]
    [Google Scholar]
  2. Amorim JH, Alves RP, Boscardin SB, Ferreira LC. The dengue virus non-structural 1 protein: risks and benefits. Virus Res 2014;181:53–60 [CrossRef][PubMed]
    [Google Scholar]
  3. Somnuke P, Hauhart RE, Atkinson JP, Diamond MS, Avirutnan P. N-linked glycosylation of dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement. Virology 2011;413:253–264 [CrossRef][PubMed]
    [Google Scholar]
  4. Falgout B, Markoff L. Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic reticulum. J Virol 1995;69:7232–7243[PubMed]
    [Google Scholar]
  5. Smith GW, Wright PJ. Synthesis of proteins and glycoproteins in dengue type 2 virus-infected vero and Aedes albopictus cells. J Gen Virol 1985;66:559–571 [CrossRef][PubMed]
    [Google Scholar]
  6. Westaway EG, Goodman MR. Variation in distribution of the three flavivirus-specified glycoproteins detected by immunofluorescence in infected Vero cells. Arch Virol 1987;94:215–228 [CrossRef][PubMed]
    [Google Scholar]
  7. Winkler G, Randolph VB, Cleaves GR, Ryan TE, Stollar V. Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer. Virology 1988;162:187–196 [CrossRef][PubMed]
    [Google Scholar]
  8. Mason PW. Maturation of Japanese encephalitis virus glycoproteins produced by infected mammalian and mosquito cells. Virology 1989;169:354–364 [CrossRef][PubMed]
    [Google Scholar]
  9. Winkler G, Maxwell SE, Ruemmler C, Stollar V. Newly synthesized dengue-2 virus nonstructural protein NS1 is a soluble protein but becomes partially hydrophobic and membrane-associated after dimerization. Virology 1989;171:302–305 [CrossRef][PubMed]
    [Google Scholar]
  10. Flamand M, Megret F, Mathieu M, Lepault J, Rey FA et al. Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol 1999;73:6104–6110[PubMed]
    [Google Scholar]
  11. Alcalá AC, Medina F, González-Robles A, Salazar-Villatoro L, Fragoso-Soriano RJ et al. The dengue virus non-structural protein 1 (NS1) is secreted efficiently from infected mosquito cells. Virology 2016;488:278–287 [CrossRef][PubMed]
    [Google Scholar]
  12. Avirutnan P, Zhang L, Punyadee N, Manuyakorn A, Puttikhunt C et al. Secreted NS1 of dengue virus attaches to the surface of cells via interactions with heparan sulfate and chondroitin sulfate. PLoS Pathog 2007;3:e183 [CrossRef][PubMed]
    [Google Scholar]
  13. Ludert JE, Mosso C, Ceballos-Olvera I, del Angel RM. Use of a commercial enzyme immunoassay to monitor dengue virus replication in cultured cells. Virol J 2008;5:51 [CrossRef][PubMed]
    [Google Scholar]
  14. Ramirez AH, Moros Z, Comach G, Zambrano J, Bravo L et al. Evaluation of dengue NS1 antigen detection tests with acute sera from patients infected with dengue virus in Venezuela. Diagn Microbiol Infect Dis 2009;65:247–253 [CrossRef][PubMed]
    [Google Scholar]
  15. Juárez-Martínez AB, Vega-Almeida TO, Salas-Benito M, García-Espitia M, De Nova-Ocampo M et al. Detection and sequencing of defective viral genomes in C6/36 cells persistently infected with dengue virus 2. Arch Virol 2013;158:583–599 [CrossRef][PubMed]
    [Google Scholar]
  16. Thiemmeca S, Tamdet C, Punyadee N, Prommool T, Songjaeng A et al. Secreted NS1 protects dengue virus from mannose-binding lectin-mediated neutralization. J Immunol 2016;197:4053–4065 [CrossRef][PubMed]
    [Google Scholar]
  17. Mackenzie JM, Jones MK, Young PR. Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication. Virology 1996;220:232–240 [CrossRef][PubMed]
    [Google Scholar]
  18. Westaway EG, Mackenzie JM, Kenney MT, Jones MK, Khromykh AA. Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures. J Virol 1997;71:6650–6661[PubMed]
    [Google Scholar]
  19. Youn S, Li T, Mccune BT, Edeling MA, Fremont DH et al. Evidence for a genetic and physical interaction between nonstructural proteins NS1 and NS4B that modulates replication of West Nile virus. J Virol 2012;86:7360–7371 [CrossRef][PubMed]
    [Google Scholar]
  20. Scaturro P, Cortese M, Chatel-Chaix L, Fischl W, Bartenschlager R. Dengue virus non-structural protein 1 modulates infectious particle production via interaction with the structural proteins. PLoS Pathog 2015;11:e1005277 [CrossRef][PubMed]
    [Google Scholar]
  21. Akey DL, Brown WC, Dutta S, Konwerski J, Jose J et al. Flavivirus NS1 structures reveal surfaces for associations with membranes and the immune system. Science 2014;343:881–885 [CrossRef][PubMed]
    [Google Scholar]
  22. Gutsche I, Coulibaly F, Voss JE, Salmon J, D'Alayer J et al. Secreted dengue virus nonstructural protein NS1 is an atypical barrel-shaped high-density lipoprotein. Proc Natl Acad Sci USA 2011;108:8003–8008 [CrossRef][PubMed]
    [Google Scholar]
  23. Cervantes-Salazar M, Angel-Ambrocio AH, Soto-Acosta R, Bautista-Carbajal P, Hurtado-Monzon AM et al. Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells. Virology 2015;484:113–126 [CrossRef][PubMed]
    [Google Scholar]
  24. Allonso D, Andrade IS, Conde JN, Coelho DR, Rocha DC et al. Dengue virus NS1 protein modulates cellular energy metabolism by increasing glyceraldehyde-3-phosphate dehydrogenase activity. J Virol 2015;89:11871–11883 [CrossRef][PubMed]
    [Google Scholar]
  25. Chuang YC, Wang SY, Lin YS, Chen HR, Yeh TM. Re-evaluation of the pathogenic roles of nonstructural protein 1 and its antibodies during dengue virus infection. J Biomed Sci 2013;20:42 [CrossRef][PubMed]
    [Google Scholar]
  26. Jacobs MG, Robinson PJ, Bletchly C, Mackenzie JM, Young PR. Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction. Faseb J 2000;14:1603–1610 [CrossRef][PubMed]
    [Google Scholar]
  27. Alcon-Lepoder S, Drouet MT, Roux P, Frenkiel MP, Arborio M et al. The secreted form of dengue virus nonstructural protein NS1 is endocytosed by hepatocytes and accumulates in late endosomes: implications for viral infectivity. J Virol 2005;79:11403–11411 [CrossRef][PubMed]
    [Google Scholar]
  28. Avirutnan P, Fuchs A, Hauhart RE, Somnuke P, Youn S et al. Antagonism of the complement component C4 by flavivirus nonstructural protein NS1. J Exp Med 2010;207:793–806 [CrossRef][PubMed]
    [Google Scholar]
  29. Avirutnan P, Hauhart RE, Somnuke P, Blom AM, Diamond MS et al. Binding of flavivirus nonstructural protein NS1 to C4b binding protein modulates complement activation. J Immunol 2011;187:424–433 [CrossRef][PubMed]
    [Google Scholar]
  30. Conde JN, da Silva EM, Allonso D, Coelho DR, Andrade ID et al. Inhibition of the membrane attack complex by Dengue virus NS1 through interaction with vitronectin and terminal complement proteins. J Virol 2016;90:9570–9581 [CrossRef][PubMed]
    [Google Scholar]
  31. Beatty PR, Puerta-Guardo H, Killingbeck SS, Glasner DR, Hopkins K et al. Dengue virus NS1 triggers endothelial permeability and vascular leak that is prevented by NS1 vaccination. Sci Transl Med 2015;7:304ra141 [CrossRef][PubMed]
    [Google Scholar]
  32. Puerta-Guardo H, Glasner DR, Harris E. Dengue virus NS1 disrupts the endothelial glycocalyx, leading to hyperpermeability. PLoS Pathog 2016;12:e1005738 [CrossRef][PubMed]
    [Google Scholar]
  33. Modhiran N, Watterson D, Muller DA, Panetta AK, Sester DP et al. Dengue virus NS1 protein activates cells via toll-like receptor 4 and disrupts endothelial cell monolayer integrity. Sci Transl Med 2015;7:304ra142 [CrossRef][PubMed]
    [Google Scholar]
  34. Libraty DH, Young PR, Pickering D, Endy TP, Kalayanarooj S et al. High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis 2002;186:1165–1168 [CrossRef][PubMed]
    [Google Scholar]
  35. De La Cruz-Hernández SI, Flores-Aguilar H, González-Mateos S, López-Martinez I, Alpuche-Aranda C et al. Determination of viremia and concentration of circulating nonstructural protein 1 in patients infected with dengue virus in Mexico. Am J Trop Med Hyg 2013;88:446–454 [CrossRef][PubMed]
    [Google Scholar]
  36. Falconar AK. The dengue virus nonstructural-1 protein (NS1) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis. Arch Virol 1997;142:897–916 [CrossRef][PubMed]
    [Google Scholar]
  37. Sun DS, Chang YC, Lien TS, King CC, Shih YL et al. Endothelial cell sensitization by death receptor fractions of an anti-dengue nonstructural protein 1 antibody induced plasma leakage, coagulopathy, and mortality in mice. J Immunol 2015;195:2743–2753 [CrossRef][PubMed]
    [Google Scholar]
  38. Wan SW, Yang YW, Chu YT, Lin CF, Chang CP et al. Anti-dengue virus nonstructural protein 1 antibodies contribute to platelet phagocytosis by macrophages. Thromb Haemost 2016;115:646–656 [CrossRef][PubMed]
    [Google Scholar]
  39. Schlesinger JJ, Brandriss MW, Walsh EE. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol 1987;68:853–857 [CrossRef][PubMed]
    [Google Scholar]
  40. Nebenführ A, Ritzenthaler C, Robinson DG. Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol 2002;130:1102–1108 [CrossRef][PubMed]
    [Google Scholar]
  41. Sreenivasan V, Ng KL, Mah Lee Ng. Brefeldin A affects West Nile virus replication in Vero cells but not C6/36 cells. J Virol Meth 1993;45:1–17 [CrossRef]
    [Google Scholar]
  42. Pierson TC, Diamond MS. Degrees of maturity: the complex structure and biology of flaviviruses. Curr Opin Virol 2012;2:168–175 [CrossRef][PubMed]
    [Google Scholar]
  43. Cutrona MB, Beznoussenko GV, Fusella A, Martella O, Moral P et al. Silencing of mammalian Sar1 isoforms reveals COPII-independent protein sorting and transport. Traffic 2013;14:691–708 [CrossRef][PubMed]
    [Google Scholar]
  44. Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 2002;54:431–467 [CrossRef][PubMed]
    [Google Scholar]
  45. Urbani L, Simoni RD. Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane. J Biol Chem 1990;265:1919–1923[PubMed]
    [Google Scholar]
  46. Kaplan MR, Simoni RD. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane. J Cell Biol 1985;101:446–453 [CrossRef][PubMed]
    [Google Scholar]
  47. Noisakran S, Dechtawewat T, Avirutnan P, Kinoshita T, Siripanyaphinyo U et al. Association of dengue virus NS1 protein with lipid rafts. J Gen Virol 2008;89:2492–2500 [CrossRef][PubMed]
    [Google Scholar]
  48. Hsieh P, Robbins PW. Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopictus mosquito cells. J Biol Chem 1984;259:2375–2382[PubMed]
    [Google Scholar]
  49. Byrne DP, Dart C, Rigden DJ. Evaluating caveolin interactions: do proteins interact with the caveolin scaffolding domain through a widespread aromatic residue-rich motif?. PLoS One 2012;7:e44879 [CrossRef][PubMed]
    [Google Scholar]
  50. Rui H, Root KT, Lee J, Glover KJ, Im W. Probing the U-shaped conformation of caveolin-1 in a bilayer. Biophys J 2014;106:1371–1380 [CrossRef][PubMed]
    [Google Scholar]
  51. Nickel W, Rabouille C. Mechanisms of regulated unconventional protein secretion. Nat Rev Mol Cell Biol 2009;10:148–155 [CrossRef][PubMed]
    [Google Scholar]
  52. Zhang M, Zeng CQ, Morris AP, Estes MK. A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells. J Virol 2000;74:11663–11670 [CrossRef][PubMed]
    [Google Scholar]
  53. Bugarcic A, Taylor JA. Rotavirus nonstructural glycoprotein NSP4 is secreted from the apical surfaces of polarized epithelial cells. J Virol 2006;80:12343–12349 [CrossRef][PubMed]
    [Google Scholar]
  54. Didsbury A, Wang C, Verdon D, Sewell MA, Mcintosh JD et al. Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells. Virol J 2011;8:551 [CrossRef][PubMed]
    [Google Scholar]
  55. Zhang L, Li D, Xu R, Zheng S, He H et al. Structural and functional analyses of a sterol carrier protein in Spodoptera litura. PLoS One 2014;9:e81542 [CrossRef][PubMed]
    [Google Scholar]
  56. Soto-Acosta R, Mosso C, Cervantes-Salazar M, Puerta-Guardo H, Medina F et al. The increase in cholesterol levels at early stages after dengue virus infection correlates with an augment in LDL particle uptake and HMG-CoA reductase activity. Virology 2013;442:132–147 [CrossRef][PubMed]
    [Google Scholar]
  57. García Cordero J, León Juárez M, González-Y-Merchand JA, Cedillo Barrón L, Gutiérrez Castañeda B. Caveolin-1 in lipid rafts interacts with dengue virus NS3 during polyprotein processing and replication in HMEC-1 cells. PLoS One 2014;9:e90704 [CrossRef][PubMed]
    [Google Scholar]
  58. Umashankar M, Sánchez-San Martín C, Liao M, Reilly B, Guo A et al. Differential cholesterol binding by class II fusion proteins determines membrane fusion properties. J Virol 2008;82:9245–9253 [CrossRef][PubMed]
    [Google Scholar]
  59. Ball JM, Schroeder ME, Williams CV, Schroeder F, Parr RD. Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1. Virol J 2013;10:336 [CrossRef][PubMed]
    [Google Scholar]
  60. Liu H, Yang L, Zhang Q, Mao L, Jiang H et al. Probing the structure and dynamics of caveolin-1 in a caveolae-mimicking asymmetric lipid bilayer model. Eur Biophys J 2016;45:511–521 [CrossRef][PubMed]
    [Google Scholar]
  61. Huang JH, Lu L, Lu H, Chen X, Jiang S et al. Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1. J Biol Chem 2007;282:6143–6152 [CrossRef][PubMed]
    [Google Scholar]
  62. Fridolfsson HN, Roth DM, Insel PA, Patel HH. Regulation of intracellular signaling and function by caveolin. Faseb J 2014;28:3823–3831 [CrossRef][PubMed]
    [Google Scholar]
  63. Cai QC, Jiang QW, Zhao GM, Guo Q, Cao GW et al. Putative caveolin-binding sites in SARS-CoV proteins. Acta Pharmacol Sin 2003;24:1051–1059[PubMed]
    [Google Scholar]
  64. Gould E, Clegg J. Growth, Titration and Purification of Alphaviruses and Flaviviruses. Virology: A Practical Approach Oxford, United Kingdom: IRL Press; 1991; pp.43–78
    [Google Scholar]
  65. Lan Q, Fallon AM. Small heat shock proteins distinguish between two mosquito species and confirm identity of their cell lines. Am J Trop Med Hyg 1990;43:669–676 [CrossRef][PubMed]
    [Google Scholar]
  66. Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 2006;3:995–1000 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000881
Loading
/content/journal/jgv/10.1099/jgv.0.000881
Loading

Data & Media loading...

Supplementary File 1

PDF

Most Cited This Month

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