Archives of Virology

, Volume 163, Issue 11, pp 3023–3033 | Cite as

Soft-shelled turtle iridovirus enters cells via cholesterol-dependent, clathrin-mediated endocytosis as well as macropinocytosis

  • Youhua Huang
  • Xiaohong HuangEmail author
  • Shaowen Wang
  • Yepin Yu
  • Songwei Ni
  • Qiwei QinEmail author
Original Article


Ranaviruses are nucleoplasmic large DNA viruses that can cause major economic losses in the aquaculture industry and pose a severe threat to global ecological diversity. The available literature demonstrates that classifiable members of the genus Ranavirus enter cells via multiple and complicated routes. Here, we demonstrated the underlying cellular entry mechanism of soft-shelled turtle iridovirus (STIV) using green fluorescence tagged recombinant virus. Treatment with chlorpromazine, sucrose, ethyl-isopropyl amiloride, chloroquine or bafilomycin A1 all significantly decreased STIV infection, suggesting that STIV uses clathrin-mediated endocytosis and macropinocytosis to enter cells via a pH-dependent pathway. Depletion of cellular cholesterol with methyl-β-cyclodextrin significantly inhibited STIV entry, but neither filipin III nor nystatin did, suggesting that STIV entry was cholesterol dependent but caveola independent. Treatment with dynasore, genistein, ML-7 or cytochalasin D all significantly inhibited STIV infection, indicating that Rac GTPase and myosin II activity were required for the macropinocytosis-like pathway as well as actin polymerization. Our findings suggest that the molecular events involved in STIV entry are not identical to those of other ranavirus isolates. Our results also extend our understanding of the molecular mechanism of iridovirus entry and pathogenesis.



We thank Jianlin Zhang for his help with flow cytometry analysis.


This work was supported by grants from the National Natural Science Foundation of China (31172445), National Key R&D Program of China” (2017YFC1404504), and the Knowledge Innovation Program of the Chinese Academy of Sciences (SQ201014).

Compliance with ethical standards

Conflict of interest

All the authors have no conflict of interest to declare.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. 1.
    Mercer J, Helenius A (2009) Virus entry by macropinocytosis. Nat Cell Biol 11:510–520CrossRefPubMedGoogle Scholar
  2. 2.
    Grove J, Marsh M (2011) The cell biology of receptor-mediated virus entry. J Cell Biol 195:1071–1082CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    McMahon HT, Boucrot E (2011) Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 12:517–533CrossRefPubMedGoogle Scholar
  4. 4.
    Daecke J, Fackler OT, Dittmar MT, Kräusslich HG (2005) Involvement of clathrin-mediated endocytosis in human immunodeficiency virus type 1 entry. J Virol 79:1581–1594CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Blanchard E, Belouzard S, Goueslain L, Wakita T, Dubuisson J, Wychowski C, Rouillé Y (2006) Hepatitis C virus entry depends on clathrin-mediated endocytosis. J Virol 80:6964–6972CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Huang HC, Chen CC, Chang WC, Tao MH, Huang C (2012) Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis. J Virol 86:9443–9453CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Piccinotti S, Kirchhausen T, Whelan SP (2013) Uptake of rabies virus into epithelial cells by clathrin-mediated endocytosis depends upon actin. J Virol 87:11637–11647CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Méndez E, Muñoz-Yañez C, Sánchez-San Martín C, Aguirre-Crespo G, Baños-Lara Mdel R, Gutierrez M, Espinosa R, Acevedo Y, Arias CF, López S (2014) Characterization of human astrovirus cell entry. J Virol 88:2452–2460CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sieczkarski SB, Whittaker GR (2002) Dissecting virus entry via endocytosis. J Gen Virol 83:1535–1545CrossRefPubMedGoogle Scholar
  10. 10.
    Pelkmans L, Kartenbeck J, Helenius A (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 3:473–483CrossRefPubMedGoogle Scholar
  11. 11.
    Marjomäki V, Pietiäinen V, Matilainen H, Upla P, Ivaska J, Nissinen L, Reunanen H, Huttunen P, Hyypiä T, Heino J (2002) Internalization of echovirus 1 in caveolae. J Virol 76:1856–1865CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Macovei A, Radulescu C, Lazar C, Petrescu S, Durantel D, Dwek RA, Zitzmann N, Nichita NB (2010) Hepatitis B virus requires intact caveolin-1 function for productive infection in HepaRG cells. J Virol 84:243–253CrossRefPubMedGoogle Scholar
  13. 13.
    Guo CJ, Liu D, Wu YY, Yang XB, Yang LS, Mi S, Huang YX, Luo YW, Jia KT, Liu ZY, Chen WJ, Weng SP, Yu XQ, He JG (2011) Entry of tiger frog virus (an Iridovirus) into HepG2 cells via a pH-dependent, atypical, caveola-mediated endocytosis pathway. J Virol 85:6416–6426CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Mercer J, Helenius A (2012) Gulping rather than sipping: macropinocytosis as a way of virus entry. Curr Opin Microbiol 15:490–499CrossRefPubMedGoogle Scholar
  15. 15.
    Krzyzaniak MA, Zumstein MT, Gerez JA, Picotti P, Helenius A (2013) Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein. PLoS Pathog 9:e1003309CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Saeed MF, Kolokoltsov AA, Albrecht T, Davey RA (2010) Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes. PLoS Pathog 6:e1001110CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wang S, Huang X, Huang Y, Hao X, Xu H, Cai M, Wang H, Qin Q (2014) A novel marine DNA virus (Singapore grouper iridovirus, SGIV) entry into host cells occurs via clathrin-mediated endocytosis and macropinocytosis in a pH-dependent manner. J Virol 88:13047–13063CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Chinchar VG (2002) Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Arch Virol 147:447–470CrossRefPubMedGoogle Scholar
  19. 19.
    Zhu YQ, Wang XL (2016) Genetic diversity of ranaviruses in amphibians in China: 10 new isolates and their implications. Pak J. Zool 48:107–114Google Scholar
  20. 20.
    Braunwald J, Nonnenmacher H, Tripier-Darcy F (1985) Ultrastructural and biochemical study of frog virus 3 uptake by BHK-21 cells. J Gen Virol 66:283–293CrossRefPubMedGoogle Scholar
  21. 21.
    Guo CJ, Wu YY, Yang LS, Yang XB, He J, Mi S, Jia KT, Weng SP, Yu XQ, He JG (2012) Infectious spleen and kidney necrosis virus (a fish iridovirus) enters Mandarin fish fry cells via caveola-dependent endocytosis. J Virol 86:2621–2631CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chen ZX, Zheng JC, Jiang YL (1999) A new iridovirus isolated from soft-shelled turtle. Virus Res 63:147–151CrossRefPubMedGoogle Scholar
  23. 23.
    Huang YH, Huang XH, Liu H, Gong J, Ouyang ZL, Cui HC, Cao JH, Zhao Y, Wang X, Jiang YL, Qin QW (2009) Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae. BMC Genomics 10:224CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Huang Y, Huang X, Cai J, Ye F, Qin Q (2011) Involvement of the mitogen-activated protein kinase pathway in soft-shelled turtle iridovirus-induced apoptosis. Apoptosis 16(6):581–593CrossRefPubMedGoogle Scholar
  25. 25.
    Huang Y, Huang X, Cai J, Ye F, Guan L, Liu H, Qin Q (2011) Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication. Virus Res 160(1–2):221–229CrossRefPubMedGoogle Scholar
  26. 26.
    Brandenburg B, Zhuang X (2007) Virus trafficking—learning from single-virus tracking. Nat Rev Microbiol 5:197–208CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    DeWire SM, Money ES, Krall SP, Damania B (2003) Rhesusmonkeyrhadinovirus (RRV): construction of a RRV-GFP recombinant virus and development of assays to assess viral replication. Virology 312:122–134CrossRefPubMedGoogle Scholar
  28. 28.
    Wang H, Yang P, Liu K, Guo F, Zhang Y, Zhang G, Jiang C (2008) SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res 18:290–301CrossRefPubMedGoogle Scholar
  29. 29.
    Chen CL, Hou WH, Liu IH, Hsiao G, Huang SS, Huang JS (2009) Inhibitors of clathrin-dependent endocytosis enhance TGFbeta signaling and responses. J Cell Sci 122:1863–1871CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Nomura R, Kiyota A, Suzaki E, Kataoka K, Ohe Y, Miyamoto K, Senda T, Fujimoto T (2004) Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae. J Virol 78:8701–8708CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zuhorn IS, Kalicharan Hoekstra D (2002) Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis. J Biol Chem 277:18021–18028CrossRefPubMedGoogle Scholar
  32. 32.
    Vela EM, Zhang L, Colpitts TM, Davey RA, Aronson JF (2007) Arenavirus entry occurs through a cholesterol-dependent, non-caveolar, clathrin-mediated endocytic mechanism. Virology 369:1–11CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Mayor S, Pagano RE (2007) Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8:603–612CrossRefPubMedGoogle Scholar
  34. 34.
    Marsh M, Helenius A (2006) Virus entry: open sesame. Cell 124:729–740CrossRefPubMedGoogle Scholar
  35. 35.
    Raghu H, Sharma-Walia N, Veettil MV, Sadagopan S, Chandran B (2009) Kaposi’s sarcoma-associated herpesvirus utilizes an actin polymerization-dependent macropinocytic pathway to enter human dermal microvascular endothelial and human umbilical vein endothelial cells. J Virol 83:4895–4911CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Mercer J, Helenius A (2008) Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 320:531–535CrossRefPubMedGoogle Scholar
  37. 37.
    Sánchez EG, Quintas A, Pérez-Núñez D, Nogal M, Barroso S, Carrascosa ÁL, Revilla Y (2012) African swine fever virus uses macropinocytosis to enter host cells. PLoS Pathog 8:e1002754CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Nanbo A, Imai M, Watanabe S, Noda T, Takahashi K, Neumann G, Halfmann P, Kawaoka Y (2010) Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog 23:e1001121CrossRefGoogle Scholar
  39. 39.
    Haspot F, Lavault A, Sinzger C, Laib Sampaio K, Stierhof YD, Pilet P, Bressolette-Bodin C, Halary F (2012) Human cytomegalovirus entry into dendritic cells occurs via a macropinocytosis-like pathway in a pH-independent and cholesterol-dependent manner. PLoS One 7:e34795CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    de Vries E, Tscherne DM, Wienholts MJ, Cobos-Jiménez V, Scholte F, García-Sastre A, Rottier PJ, de Haan CA (2011) Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway. PLoS Pathog 7:e1001329CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.College of Marine SciencesSouth China Agricultural UniversityGuangzhouPeople’s Republic of China
  2. 2.Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of OceanologyChinese Academy of SciencesGuangzhouPeople’s Republic of China
  3. 3.University of Chinese Academy of SciencesBeijingPeople’s Republic of China

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