Endosomes and Microtubles are Required for Productive Infection in Aquareovirus

Abstract

Grass carp reovirus (GCRV), the genus Aquareovirus in family Reoviridae, is viewed as the most pathogenic aquareovirus. To understand the molecular mechanism of how aquareovirus initiates productive infection, the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots (QDs)-tracking in combination with biochemical approaches. We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl, chloroquine and bafilomycin A1 (Bafi). Confocal images indicated that GCRV particles could colocalize with Rab5, Rab7 and lysosomes in host cells. Further ultrastructural examination validated that viral particle was found in late endosomes. Moreover, disruption of microtubules with nocodazole clearly blocked GCRV entry, while no inhibitory effects were observed with cytochalasin D treated cells in viral infection, hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament. Notably, viral particles were observed to transport along microtubules by using QD-labeled GCRV. Altogether, our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  1. Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013–2016

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. Chandran K, Walker SB, Chen Y, Contreras CM, Schiff LA, Baker TS, Nibert ML (1999) In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins μ1 and ς3. J Virol 73:3941–3950

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Chandran K, Farsetta DL, Nibert ML (2002) Strategy for nonenveloped virus entry: a hydrophobic conformer of the reovirus membrane penetration protein μ1 mediates membrane disruption. J Virol 76:9920–9933

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  4. Chen Q, Guo H, Zhang F, Fang Q (2018) N-terminal myristoylated VP5 is required for penetrating cell membrane and promoting infectivity in aquareoviruses. Virol Sin 33:287–290

    PubMed  PubMed Central  Article  Google Scholar 

  5. Doherty GJ, McMahon HT (2009) Mechanisms of endocytosis. Annu Rev Biochem 78:857–902

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. Eilers U, Klumperman J, Hauri H-P (1989) Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2). J Cell Biol 108:13–22

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. Fan C, Shao L, Fang Q (2010) Characterization of the nonstructural protein NS80 of grass carp reovirus. Arch Virol 155:1755–1763

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. Fang Q, Ke L, Cai Y (1989) Growth characterization and high titre culture of GCHV. Virol Sin 4:315–319

    Google Scholar 

  9. Fang Q, Shah S, Liang Y, Zhou H (2005) 3D reconstruction and capsid protein characterization of grass carp reovirus. Sci China C Life Sci 48:593–600

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  10. Fang Q, Seng E, Ding Q, Zhang L (2008) Characterization of infectious particles of grass carp reovirus by treatment with proteases. Arch Virol 153:675–682

    CAS  PubMed  Article  Google Scholar 

  11. Forzan M, Marsh M, Roy P (2007) Bluetongue virus entry into cells. J Virol 81:4819–4827

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Furlong DB, Nibert M, Fields B (1988) Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles. J Virol 62:246–256

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Guo H, Sun X, Yan L, Shao L, Fang Q (2013) The NS16 protein of aquareovirus-C is a fusion-associated small transmembrane (FAST) protein, and its activity can be enhanced by the nonstructural protein NS26. Virus Res 171:129–137

    CAS  PubMed  Article  Google Scholar 

  14. Huang WR, Wang YC, Chi PI, Wang L, Wang CY, Lin CH, Liu HJ (2011) Cell entry of avian reovirus follows a caveolin-1-mediated and dynamin-2-dependent endocytic pathway that requires activation of p38 mitogen-activated protein kinase (MAPK) and Src signaling pathways as well as microtubules and small GTPase Rab5 protein. J Biol Chem 286:30780–30794

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Jaafar FM, Goodwin AE, Belhouchet M, Merry G, Fang Q, Cantaloube J-F, Biagini P, de Micco P, Mertens PP, Attoui H (2008) Complete characterisation of the American grass carp reovirus genome (genus Aquareovirus: family Reoviridae) reveals an evolutionary link between aquareoviruses and coltiviruses. Virology 373:310–321

    Article  CAS  Google Scholar 

  16. Ke L, Fang Q, Cai Y (1990) Characteristics of a novel isolate of grass carp haemorrhagic virus. Acta Hydrobiol Sin 14:153–159

    Google Scholar 

  17. King AM, Lefkowitz E, Adams MJ, Carstens EB (2011) Virus taxonomy: 9th report of the international committee on taxonomy of viruses vol 9, Elsevier

  18. Li X, Fang Q (2013) High-resolution 3D structures reveal the biological functions of reoviruses. Virol Sin 28:318–325

    CAS  PubMed  Article  Google Scholar 

  19. Liu H-J, Lin P-Y, Wang L-R, Hsu H-Y, Liao M-H, Shih W-L (2008) Activation of small GTPases RhoA and Rac1 is required for avian reovirus p10-induced syncytium formation. Mol Cell 26:396–403

    CAS  Google Scholar 

  20. Liu S-L, Zhang Z-L, Tian Z-Q, Zhao H-S, Liu H, Sun E-Z, Xiao GF, Zhang W, Wang H-Z, Pang D-W (2011) Effectively and efficiently dissecting the infection of influenza virus by quantum-dot-based single-particle tracking. ACS Nano 6:141–150

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. Lopez S, Arias C (2010) How viruses hijack endocytic machinery. Nat Educ 3:16–23

    Google Scholar 

  22. Mabit H, Nakano MY, Prank U, Saam B, Döhner K, Sodeik B, Greber UF (2002) Intact microtubules support adenovirus and herpes simplex virus infections. J Virol 76:9962–9971

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Maginnis MS, Mainou BA, Derdowski A, Johnson EM, Zent R, Dermody TS (2008) NPXY motifs in the β1 integrin cytoplasmic tail are required for functional reovirus entry. J Virol 82:3181–3191

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. Mainou BA, Dermody TS (2011) SRC kinase mediates productive endocytic sorting of reovirus during cell entry. J Virol 85:3203–3213

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Mainou BA, Dermody TS (2012) Transport to late endosomes is required for efficient reovirus infection. J Virol 86:8346–8358

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Mainou BA, Zamora PF, Ashbrook AW, Dorset DC, Kim KS, Dermody TS (2013) Reovirus cell entry requires functional microtubules. MBio 4:e00405–e00413

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  27. McDonald D, Vodicka MA, Lucero G, Svitkina TM, Borisy GG, Emerman M, Hope TJ (2002) Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159:441–452

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Medintz IL, Uyeda HT, Goldman ER, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4:435

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. Nicola AV, Aguilar HC, Mercer J, Ryckman B, Wiethoff CM (2013) Virus entry by endocytosis. Adv Virol. https://doi.org/10.1155/2013/469538

    Article  PubMed  PubMed Central  Google Scholar 

  30. Patel A, Mohl B-P, Roy P (2016) Entry of bluetongue virus capsid requires the late endosome-specific lipid lysobisphosphatidic acid. J Biol Chem 291:12408–12419

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Rangel AA, Rockemann DD, Hetrick FM, Samal SK (1999) Identification of grass carp haemorrhage virus as a new genogroup of aquareovirus. J General Virol 80:2399–2402

    CAS  Article  Google Scholar 

  32. Rao Y, Su J (2015) Insights into the antiviral immunity against grass carp (Ctenopharyngodon idella) reovirus (GCRV) in grass carp. J Immunol Res. https://doi.org/10.1155/2015/670437

    Article  PubMed  PubMed Central  Google Scholar 

  33. Salsman J, Top D, Boutilier J, Duncan R (2005) Extensive syncytium formation mediated by the reovirus FAST proteins triggers apoptosis-induced membrane instability. J Virol 79:8090–8100

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 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 Pathogens 8:e1002754

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  35. Schulz WL, Haj AK, Schiff LA (2012) Reovirus uses multiple endocytic pathways for cell entry. J Virol 86:12665–12675

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. Shao L, Guo H, Yan L-M, Liu H, Fang Q (2013) Aquareovirus NS80 recruits viral proteins to its inclusions, and its C-terminal domain is the primary driving force for viral inclusion formation. PLoS ONE 8:e55334

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. Silverstein SC, Astell C, Levin DH, Schonberg M, Acs G (1972) The mechanisms of reovirus uncoating and gene activation in vivo. Virology 47:797–806

    CAS  PubMed  Article  Google Scholar 

  38. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513

    CAS  PubMed  Article  Google Scholar 

  39. Sturzenbecker L, Nibert M, Furlong D, Fields B (1987) Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle. J Virol 61:2351–2361

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Suomalainen M, Greber UF (2013) Uncoating of non-enveloped viruses. Curr Opin Virol 3:27–33

    CAS  PubMed  Article  Google Scholar 

  41. Wang Q, Zeng W, Liu C, Zhang C, Wang Y, Shi C, Wu S (2012) Complete genome sequence of a reovirus isolated from grass carp, indicating different genotypes of GCRV in China. J Virol 86:12466

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Yan L, Zhang J, Guo H, Yan S, Chen Q, Zhang F, Fang Q (2015) Aquareovirus NS80 initiates efficient viral replication by retaining core proteins within replication-associated viral inclusion bodies. PLoS ONE 10:e0126127

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  43. Yan S, Zhang J, Guo H, Yan L, Chen Q, Zhang F, Fang Q (2015) VP5 autocleavage is required for efficient infection by in vitro-recoated aquareovirus particles. J Gen Virol 96:1795–1800

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  44. Zhang F, Zheng Z, Liu S-L, Lu W, Zhang Z, Zhang C, Zhou P, Zhang Y, Long G, He Z (2013) Self-biotinylation and site-specific double labeling of baculovirus using quantum dots for single-virus in situ tracking. Biomaterials 34:7506–7518

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. Zhang F, Yan S, Guo H, Chen Q, Fang Q (2017) Characterization of viral entry and infection of quantum dot-labeled grass carp reovirus. Virol Sin 32:163–166

    PubMed  PubMed Central  Article  Google Scholar 

  46. Zhang F, Guo H, Zhang J, Chen Q, Fang Q (2018) Identification of the caveolae/raft-mediated endocytosis as the primary entry pathway for aquareovirus. Virology 513:195–207

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Prof. Hanzhong Wang for providing plasmids encoding Rab5, Rab7, actin and MAP4; Ding Gao, Anna Du and Pei Zhang for ultrathin section preparation and EM observation. We also thank the support from “Center for Instrumental Analysis and Metrology, the Core Facility and Technical Support, Wuhan Institute of Virology”. This work is supported in part by grants from the National Natural Science Foundation of China (31672693, 31972838 and 31400139, 31372565).

Author information

Affiliations

Authors

Contributions

QF designed the experiments. FZ, GH, QC, ZR and QF carried out the experiments and analyzed the data. FZ and QF wrote the paper. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Qin Fang.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

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

Electronic Supplementary Material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, F., Guo, H., Chen, Q. et al. Endosomes and Microtubles are Required for Productive Infection in Aquareovirus. Virol. Sin. 35, 200–211 (2020). https://doi.org/10.1007/s12250-019-00178-1

Download citation

Keywords

  • Aquareovirus
  • Cell entry
  • Quantum dot
  • Endosome
  • Microtubule