Archives of Virology

, Volume 159, Issue 5, pp 1005–1015 | Cite as

Understanding internalization of rotavirus VP6 nanotubes by cells: towards a recombinant vaccine

  • Mabel Rodríguez
  • Christopher Wood
  • Rosana Sanchez-López
  • Ricardo M. Castro-Acosta
  • Octavio T. Ramírez
  • Laura A. Palomares
Original Article

Abstract

Rotavirus VP6 nanotubes are an attractive option for a recombinant vaccine against rotavirus disease. Protection against rotavirus infection and an adjuvant effect have been observed upon immunization with VP6 nanotubes. However, little information exists on how VP6 nanotubes interact with cells and trigger an immune response. In this work, the interaction between VP6 nanotubes and different cell lines was characterized. VP6 nanotubes were not cytotoxic to any of the animal or human cell lines tested. Uptake of nanotubes into cells was cell-line-dependent, as only THP1 and J774 macrophage cells internalized them. Moreover, the size and spatial arrangement of VP6 assembled into nanotubes allowed their uptake by macrophages, as double-layered rotavirus-like particles also displaying VP6 in their surface were not taken up. The internalization of VP6 nanotubes was inhibited by methyl-β-cyclodextrin, but not by genistein, indicating that nanotube entry is specific, depends on the presence of cholesterol in the plasma membrane, and does not require the activity of tyrosine kinases. The information generated here expands our understanding of the interaction of protein nanotubes with cells, which is useful for the application of VP6 nanotubes as a vaccine.

Supplementary material

705_2013_1916_MOESM1_ESM.ppt (860 kb)
Supplementary material 1 (PPT 859 kb)

References

  1. 1.
    Wardlaw T, Salama P, Brocklehurst C, Chopra M, Mason E (2010) Diarrhoea: why children are still dying and what can be done. Lancet 375(9718):870–872. doi:10.1016/S0140-6736(09)61798-0 PubMedCrossRefGoogle Scholar
  2. 2.
    Rotavirus vaccines WHO position paper: January 2013—Recommendations (2013). Vaccine. doi:10.1016/j.vaccine.2013.05.037
  3. 3.
    Ward RL, McNeal MM, Steele AD (2008) Why does the world need another rotavirus vaccine? Ther Clin Risk Manag 4(1):49–63PubMedCentralPubMedGoogle Scholar
  4. 4.
    Esquivel FR, Lopez S, Guitierrez XL, Arias C (2000) The internal rotavirus protein VP6 primes for an enhanced neutralizing antibody response. Arch Virol 145(4):813–825PubMedCrossRefGoogle Scholar
  5. 5.
    Banos DM, Lopez S, Arias CF, Esquivel FR (1997) Identification of a T-helper cell epitope on the rotavirus VP6 protein. J Virol 71(1):419–426PubMedCentralPubMedGoogle Scholar
  6. 6.
    Ward RL, McNeal MM (2010) VP6: a candidate rotavirus vaccine. J Infect Dis 202(Suppl):S101–S107. doi:10.1086/653556 PubMedCrossRefGoogle Scholar
  7. 7.
    Schwartz-Cornil I, Benureau Y, Greenberg H, Hendrickson BA, Cohen J (2002) Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins. J Virol 76(16):8110–8117PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Corthesy B, Benureau Y, Perrier C, Fourgeux C, Parez N, Greenberg H, Schwartz-Cornil I (2006) Rotavirus anti-VP6 secretory immunoglobulin A contributes to protection via intracellular neutralization but not via immune exclusion. J Virol 80(21):10692–10699. doi:10.1128/JVI.00927-06 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    McNeal MM, VanCott JL, Choi AH, Basu M, Flint JA, Stone SC, Clements JD, Ward RL (2002) CD4 T cells are the only lymphocytes needed to protect mice against rotavirus shedding after intranasal immunization with a chimeric VP6 protein and the adjuvant LT(R192G). J Virol 76(2):560–568PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Smiley KL, McNeal MM, Basu M, Choi AH, Clements JD, Ward RL (2007) Association of gamma interferon and interleukin-17 production in intestinal CD4+ T cells with protection against rotavirus shedding in mice intranasally immunized with VP6 and the adjuvant LT(R192G). J Virol 81(8):3740–3748. doi:10.1128/JVI.01877-06 PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Lepault J, Petitpas I, Erk I, Navaza J, Bigot D, Dona M, Vachette P, Cohen J, Rey FA (2001) Structural polymorphism of the major capsid protein of rotavirus. Embo J 20(7):1498–1507. doi:10.1093/emboj/20.7.1498 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Ready KFM, Sabara M (1987) Invitro assembly of bovine rotavirus nucleocapsid protein. Virology 157(1):189–198PubMedCrossRefGoogle Scholar
  13. 13.
    Blazevic V, Lappalainen S, Nurminen K, Huhti L, Vesikari T (2011) Norovirus VLPs and rotavirus VP6 protein as combined vaccine for childhood gastroenteritis. Vaccine 29(45):8126–8133. doi:10.1016/j.vaccine.2011.08.026 PubMedCrossRefGoogle Scholar
  14. 14.
    Pastor-Flores AR Rodríguez-Limas WA, Contreras MA, Esquivel-Soto E, Esquivel-Guadarrama F, Ramírez OT, Palomares LA (2013) The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (in press)Google Scholar
  15. 15.
    Ghosh MK, Borca MV, Roy P (2002) Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses. Virology 302(2):383–392 (pii:S004268220291648X)PubMedCrossRefGoogle Scholar
  16. 16.
    Mathieu M, Petitpas I, Navaza J, Lepault J, Kohli E, Pothier P, Prasad BV, Cohen J, Rey FA (2001) Atomic structure of the major capsid protein of rotavirus: implications for the architecture of the virion. Embo J 20(7):1485–1497. doi:10.1093/emboj/20.7.1485 PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Geng Y, Dalhaimer P, Cai S, Tsai R, Tewari M, Minko T, Discher DE (2007) Shape effects of filaments versus spherical particles in flow and drug delivery. Nat Nanotechnol 2(4):249–255. doi:10.1038/nnano.2007.70 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8(12):958–969. doi:10.1038/nri2448 PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Plascencia-Villa G, Mena JA, Castro-Acosta RM, Fabian JC, Ramirez OT, Palomares LA (2011) Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials. J Chromatogr B 879(15–16):1105–1111. doi:10.1016/j.jchromb.2011.03.027 CrossRefGoogle Scholar
  20. 20.
    Castro-Acosta RM, Revilla AL, Ramirez OT, Palomares LA (2010) Separation and quantification of double- and triple-layered rotavirus-like particles by CZE. Electrophoresis 31(8):1376–1381. doi:10.1002/elps.200900558 PubMedCrossRefGoogle Scholar
  21. 21.
    Vercauteren D, Vandenbroucke RE, Jones AT, Rejman J, Demeester J, De Smedt SC, Sanders NN, Braeckmans K (2010) The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol Ther 18(3):561–569. doi:10.1038/Mt.2009.281 PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Greenberg HB, Valdesuso J, Vanwyke K, Midthun K, Walsh M, Mcauliffe V, Wyatt RG, Kalica AR, Flores J, Hoshino Y (1983) Production and preliminary characterization of monoclonal-antibodies directed at 2 surface-proteins of rhesus rotavirus. J Virol 47(2):267–275PubMedCentralPubMedGoogle Scholar
  23. 23.
    Mena JA, Ramirez OT, Palomares LA (2006) Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system. J Biotechnol 122(4):443–452. doi:10.1016/j.jbiotec.2005.10.005 PubMedCrossRefGoogle Scholar
  24. 24.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. doi:10.1038/nmeth.2019 PubMedCrossRefGoogle Scholar
  25. 25.
    Plascencia-Villa G, Saniger JM, Ascencio JA, Palomares LA, Ramirez OT (2009) Use of recombinant rotavirus VP6 nanotubes as a multifunctional template for the synthesis of nanobiomaterials functionalized with metals. Biotechnol Bioeng 104(5):871–881. doi:10.1002/bit.22497 PubMedCrossRefGoogle Scholar
  26. 26.
    Mrakovcic M, Absenger M, Riedl R, Smole C, Roblegg E, Frohlich LF, Frohlich E (2013) Assessment of long-term effects of nanoparticles in a microcarrier cell culture system. Plos One 8(2):e56791. doi:10.1371/journal.pone.0056791 PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Kagan BL, Jang H, Capone R, Teran Arce F, Ramachandran S, Lal R, Nussinov R (2012) Antimicrobial properties of amyloid peptides. Mol Pharm 9(4):708–717. doi:10.1021/mp200419b PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Harrison RS, Sharpe PC, Singh Y, Fairlie DP (2007) Amyloid peptides and proteins in review. Rev Physiol Biochem Pharmacol 159:1–77. doi:10.1007/112_2007_0701 PubMedGoogle Scholar
  29. 29.
    Fifis T, Gamvrellis A, Crimeen-Irwin B, Pietersz GA, Li J, Mottram PL, McKenzie IF, Plebanski M (2004) Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol 173(5):3148–3154 (pii:173/5/3148)PubMedCrossRefGoogle Scholar
  30. 30.
    Jones AT (2008) Gateways and tools for drug delivery: endocytic pathways and the cellular dynamics of cell penetrating peptides. Int J Pharm 354(1–2):34–38. doi:10.1016/j.ijpharm.2007.10.046 PubMedCrossRefGoogle Scholar
  31. 31.
    Gualtero DF, Guzman F, Acosta O, Guerrero CA (2007) Amino acid domains 280-297 of VP6 and 531-554 of VP4 are implicated in heat shock cognate protein hsc70-mediated rotavirus infection. Arch Virol 152(12):2183–2196. doi:10.1007/s00705-007-1055-5 PubMedCrossRefGoogle Scholar
  32. 32.
    Triantafilou K, Triantafilou M, Dedrick RL (2001) A CD14-independent LPS receptor cluster. Nat Immunol 2(4):338–345. doi:10.1038/86342 PubMedCrossRefGoogle Scholar
  33. 33.
    Arnold-Schild D, Hanau D, Spehner D, Schmid C, Rammensee HG, de la Salle H, Schild H (1999) Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells. J Immunol 162(7):3757–3760PubMedGoogle Scholar
  34. 34.
    Foster LJ, De Hoog CL, Mann M (2003) Unbiased quantitative proteomics of lipid rafts reveals high specificity for signaling factors. Proc Natl Acad Sci USA 100(10):5813–5818. doi:10.1073/pnas.0631608100 PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Yeung T, Grinstein S (2007) Lipid signaling and the modulation of surface charge during phagocytosis. Immunol Rev 219:17–36PubMedCrossRefGoogle Scholar
  36. 36.
    Botelho RJ, Teruel M, Dierckman R, Anderson R, Wells A, York JD, Meyer T, Grinstein S (2000) Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J Cell Biol 151(7):1353–1367PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Falkenburger BH, Jensen JB, Dickson EJ, Suh BC, Hille B (2010) Phosphoinositides: lipid regulators of membrane proteins. J Physiol London 588(17):3179–3185. doi:10.1113/jphysiol.2010.192153 PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Nagao G, Ishii K, Hirota K, Makino K, Terada H (2010) Role of lipid rafts in phagocytic uptake of polystyrene latex microspheres by macrophages. Anticancer Res 30(8):3167–3176PubMedGoogle Scholar
  39. 39.
    Gatfield J, Pieters J (2000) Essential role for cholesterol in entry of mycobacteria into macrophages. Science 288(5471):1647–1650PubMedCrossRefGoogle Scholar
  40. 40.
    Fra AM, Williamson E, Simons K, Parton RG (1995) De-novo formation of caveolae in lymphocytes by expression of Vip21-caveolin. Proc Natl Acad Sci USA 92(19):8655–8659PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2013

Authors and Affiliations

  • Mabel Rodríguez
    • 1
  • Christopher Wood
    • 2
  • Rosana Sanchez-López
    • 3
  • Ricardo M. Castro-Acosta
    • 1
  • Octavio T. Ramírez
    • 1
  • Laura A. Palomares
    • 1
  1. 1.Departamento de Medicina Molecular y Bioprocesos, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  3. 3.Departamento de Biología Molecular de Plantas, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

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