Virologica Sinica

, Volume 32, Issue 3, pp 207–215

Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells

  • Waqas Ahmad
  • Yingying Li
  • Yidi Guo
  • Xinyu Wang
  • Ming Duan
  • Zhenhong Guan
  • Zengshan Liu
  • Maolin Zhang
Research Article


Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.


Rab5 Rab7 rabies virus(RABV) endosomes colocalization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Barbieri MA, Roberts RL, Gumusboga A, Highfield H, Alvarez- Dominguez C, Wells A, Stahl PD. 2000. Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5. J Cell Biol, 151: 539–550.PubMedGoogle Scholar
  2. Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M. 1992. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell, 70: 715–728.CrossRefPubMedGoogle Scholar
  3. Callaghan J, Simonsen A, GaullierJM, Ban-Hock TO, Stenmark H. 1999. The endosome fusion regulator early-endosomal autoantigen 1 (EEA1) is a dimer. Biochem J, 338: 539–543.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Caviston JP, Holzbaur EL. 2006. Microtubule motors at the intersection of trafficking and transport. Trends Cell Biol, 16: 530–537.CrossRefPubMedGoogle Scholar
  5. Ceresa BP, Bahr SJ. 2006. Rab7 activity affects epidermal growth factor: epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome. J BiolChem, 281: 1099–1106.Google Scholar
  6. Cook NR, Row PE, Davidson HW. 2004. Lysosome Associated Membrane Protein 1 (Lamp1)Traffics Directly from the TGN to Early Endosomes. Traffic, 5: 685–699.CrossRefPubMedGoogle Scholar
  7. Colpitts TM, Moore AC, Kolokoltsov AA, Davey RA. 2007. Venezuelan equine encephalitis virus infection of mosquito cells requires acidification as well as mosquito homologs of the endocytic proteins Rab5 and Rab7. Virology, 369: 78–91.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Deinhardt K, Salinas S, Verastegui C, Watson R, Worth D, Hanrahan S, Bucci C, Schiavo G. 2006. Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway of Neuron. Neuron, 52: 293–305.CrossRefPubMedGoogle Scholar
  9. Flannigan DJ, Zewail AH. 2012. 4D electron microscopy: principles and applications. AccChem Res, 45: 1828–1839.CrossRefGoogle Scholar
  10. Hislop JN, Islam TA, Eleftheriadou I, Carpentier DC, Trabalza A, Parkinson M, Schiavo G, Mazarakis ND. 2014. Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons. J BiolChem, 289: 16148–16163.Google Scholar
  11. Huotari J, Helenius A. 2011. Endosome Maturation. EMBO J, 30: 3481–3500.CrossRefPubMedPubMedCentralGoogle Scholar
  12. Jackson AC. 2016. Human Rabies: a 2016 Update. Curr Infect Dis Rep, 18: 1–6.CrossRefGoogle Scholar
  13. Kaspar M, Trachsel E, Neri D. 2007. The antibody-mediated targeted delivery of interleukin-15 and GMCSF to the tumor neovasculature inhibits tumor growth and metastasis. Cancer Res, 67: 4940–4948.CrossRefPubMedGoogle Scholar
  14. Krishnan MN, Sukumaran B, Pal U, Agaisse H, Murray JL, Hodge TW, Fikrig E. Rab 5 is required for the cellular entry of dengue and West Nile viruses. J Virol, 81: 4881–4885.Google Scholar
  15. Lewis P, Lentz TL. 1998. Rabies virus entry into cultured rat hippo campalneurons. J Neurocytol, 27: 559–573.CrossRefPubMedGoogle Scholar
  16. Liu SL, Wu QM, Zhang LJ, Wang ZJ, Sun EZ, Zhang ZL, Pang DW. 2014. Three-Dimensional Tracking of Rab5 and Rab7 Associated Infection Process of Influenza Virus. Small, 22: 4746–4753.CrossRefGoogle Scholar
  17. Luzio JP, Pryor PR, Bright NA. 2007. Lysosomes: fusion and function. Nat Rev Mol Cell Biol, 8: 622–632.CrossRefPubMedGoogle Scholar
  18. Marsh M, Helenius A. 2006. Virus entry: open sesame. Cell, 24: 729–740.CrossRefGoogle Scholar
  19. Macovei A, Petrareanu C, Lazar C, Florian P, Branza-Nichita N. 2013. Regulation of hepatitis B virus infection by Rab5, Rab7, and the endolysosomal compartment. J Virol, 11: 6415–6427.CrossRefGoogle Scholar
  20. Miyazawa N, Crystal RG, Leopold PL. 2001. Adenovirus serotype 7 retention in a late endosomal compartment prior to cytosol escape is modulated by fiber protein. J Virol, 75: 1387–1400.CrossRefPubMedPubMedCentralGoogle Scholar
  21. McLauchlan H, Newell J, Morrice N, Osborne A, West M, Smythe E. 1998. A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits. CurrBiol, 8: 34–45.CrossRefGoogle Scholar
  22. McMahon HT, Boucrot E. 2011. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol, 12: 517–533.CrossRefPubMedGoogle Scholar
  23. Nielsen E, Severin F, Backer JM, Hyman AA, Zerial M. 1999. Rab5 regulates motility of early endosomes on microtubules. Nat Cell Biol, 1: 376–382.CrossRefPubMedGoogle Scholar
  24. Pereira-Leal J, Seabra M. 2001. Evolution of the Rab family of small GTP-binding proteins. J. MolBiol, 313: 889–901.CrossRefGoogle Scholar
  25. Piccinotti S, Kirchhausen T, Whelana SPJ. 2011. Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin. J Virol, 87: 11637–11647.CrossRefGoogle Scholar
  26. Pierrea CAS, Leonardb D, Corverab S, Kurt-Jonesa EA, Finberga RW. 2011. Antibodies to cell surface proteins redirect intracellular trafficking pathways. ExpMolPathol, 91: 723–732.Google Scholar
  27. Rink J, Ghigo E, YannisKalaidzidis Y, Zerial M. 2005. Rab Conversion as a Mechanism of Progression from Early to Late Endosomes. Cell, 122: 735–749.CrossRefPubMedGoogle Scholar
  28. Rubino M, Miaczynska M, Lippé R, Zerial M. 2000. Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes. J Biol- Chem, 275: 3745–3748.CrossRefPubMedGoogle Scholar
  29. Schmieg N, Menendez G, Schiavo G, Terenzioc M. 2013. Signalling endosomes in axonal transport: Travel updates on the molecular highway. Semin Cell Dev Biol: 1–13.Google Scholar
  30. Seabra M, Mules E, Hume A. 2002. RabGTPases, intracellular traffic and disease. Trends Mol Med, 8: 23–30.CrossRefPubMedGoogle Scholar
  31. Simonsen A, Lippe R, Christoforidis S, Gaullier JM, Brech A, Callaghan J, Toh BH, Murphy C, Zerial M, Stenmark H. 1998. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature, 394: 494–498.CrossRefPubMedGoogle Scholar
  32. Song Y, Jinli H, Bin Q, Yanchao L, Ye X, Ming D, Zhenhong G, Maolin Z, Liankun S. 2013. Street Rabies Virus Causes Dendritic Injury and F-actin depolymerization in the Hippocampus. J Gen Virol, 94: 276–283.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Stenmark H, Parton RG, Steele-Mortimer O, Lütcke A, Gruenberg J, Zerial M. 1994. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. The EMBO J, 13: 1287.PubMedGoogle Scholar
  34. Stenmark H. RabGTPases as coordinators of vesicle traffic. 2009. Nat Rev Mol Cell Biol, 10: 513–525.Google Scholar
  35. Vanlandingham PA, Ceresa BP. 2009. Rab7 Regulates Late Endocytic Trafficking Downstream of Multivesicular Body Biogenesis and Cargo Sequestration. J BiolChem, 284: 12110–12124.Google Scholar
  36. Vela EM, Colpitts T M, Zhang LH, Davey RA, Aronson JF. 2008. Pichinde virus is trafficked through a dynamin 2 endocytic pathway that is dependent on cellular Rab5- and Rab7-mediated endosomes. Arch Virol, 153: 1391–1396.CrossRefPubMedGoogle Scholar
  37. Verhoeven K, De Jonghe P, Coen K, Verpoorten N, Auer-Grumbach M, Kwon JM, FitzPatrick D, Schmedding E, De Vriendt E, Jacobs A. 2003. Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy. Am J Hum Genet, 72: 722–727.CrossRefPubMedPubMedCentralGoogle Scholar
  38. Vidricaire G, Tremblay MJ. 2005. Rab5 and Rab7, but Not ARF6, Govern the Early Events of HIV-1 Infection in Polarized Human Placental Cells. J Immunol, 175: 6517–6530.CrossRefPubMedGoogle Scholar
  39. Weir DL, Laing ED, Smith IL, Wang L, Broder CC. 2014. Host cell virus entry mediated by asutralian bat lyssavirus G envelop glycoprotein occurs through a clathrin-mediated endocytosic pathway that required actin and Rab5. Virol J, 11: 1–10.CrossRefGoogle Scholar
  40. Wilson JM, Hoop M, Zorzi N, Toh BH, Dotti CG, Parton RG. 2000. EEA1, a Tethering Protein of the Early Sorting Endosome, Shows a Polarized Distribution in Hippocampal Neurons, Epithelial Cells, and Fibroblasts. MolBiol Cell, 11: 2657–2671.Google Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Nature Singapore Pte Ltd. 2017

Authors and Affiliations

  1. 1.Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary MedicineJilin UniversityChangchunChina
  2. 2.Section of Epidemiology and Public HealthCollege of Veterinary and Animal SciencesJhangPakistan

Personalised recommendations