Abstract
Although mechanisms of bovine viral diarrhea virus (BVDV) entry into bovine cells have been elucidated, little is known concerning pestivirus entry and receptor usage in ovine cells. In this study, we determined the entry mechanisms of BVDV-1 and BVDV-2 in sheep fetal thymus cells. Both BVDV-1 and BVDV-2 infections were inhibited completely by chlorpromazine, β-methyl cyclodextrin, sucrose, bafilomycin A1, chloroquine, and ammonium chloride. Simultaneous presence of reducing agent and low pH resulted in marked loss of BVDV infectivity. Moreover, BVDV was unable to fuse with ovine cell membrane by the presence of reducing agent or low pH alone, while combination of both led to fusion at low efficiency. Furthermore, sheep fetal thymus cells acutely infected with BVDV-1 or BVDV-2 were found protected from heterologous BVDV infection. Taken together, our results showed for the first time that entry of both BVDV-1 and BVDV-2 into ovine cells occurred through clathrin-dependent endocytosis, endosomal acidification, and low pH-dependent fusion following an activation step, besides suggesting the involvement of a common ovine cellular receptor during attachment and entry.
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References
Blanchard E.; Belouzard S.; Goueslain L.; Wakita T.; Dubuisson J.; Wychowski C.; Rouille Y. Hepatitis C virus entry depends on clathrin-dependent endocytosis. J. Virol. 80: 6964–6972; 2006.
Chu J. J.; Ng M. L. Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J. Virol. 78: 10543–10555; 2004.
Deregt D.; Bolin S. R.; van den Hurk J.; Ridpath J. F.; Gilbert S. A. Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralizing escape mutants. Virus Res. 53: 81–90; 1998a.
Deregt D.; van Rijn P. A.; Wiens T. Y.; van den Hurk J. Monoclonal antibodies to the E2 protein of a new genotype (type 2) of bovine viral diarrhea virus define three antigenic domains involved in neutralization. Virus Res. 57: 171–181; 1998b.
Grummer B.; Grotha S.; Greiser-Wilke I. Bovine viral diarrhoea virus is internalized by clathrin-dependent receptor-mediated endocytosis. J. Vet. Med. B. 51: 427–432; 2004.
Krey T.; Thiel H. J.; Rumenapf T. Acid-resistant bovine pestivirus requires activation for pH-triggered fusion during entry. J. Virol. 79: 4191–4200; 2005.
Lecot S.; Belouzard S.; Dubuisson J.; Rouille Y. Bovine viral diarrhea virus entry is dependent on clathrin-mediated endocytosis. J. Virol. 79: 10826–10829; 2005.
Lee Y. M.; Tscherne D. M.; Yun S. I.; Frolov I.; Rice C. M. Dual mechanisms of pestiviral superinfection exclusion at entry and RNA replication. J. Virol. 79: 3231–3242; 2005.
Liang D.; Sainz I. F.; Ansari I. H.; Gil L. H. V. G.; Vassilev V.; Donis R. O. The envelope glycoprotein E2 is a determinant of cell culture tropism in ruminant pestiviruses. J. Gen. Virol. 84: 1269–1274; 2003.
Maurer K.; Krey T.; Moennig V.; Thiel H. J.; Rumenapf T. CD46 is a cellular receptor for bovine viral diarrhea virus. J. Virol. 78: 1792–1799; 2004.
Meertens L.; Bertaux C.; Dragic T. Hepatitis C virus requires a critical post internalization step and delivery to early endosomes via clathrin-coated vesicles. J. Virol. 80: 11571–11578; 2006.
Mellman I. Endocytosis and molecular sorting. Ann. Rev. Cell Dev. Biol. 12: 575–625; 1996.
Meyers G.; Thiel H. J. Molecular characterization of pestiviruses. Adv. Virus Res. 47: 53–118; 1996.
Mishra N.; Pattnaik B.; Vilcek S.; Patil S. S.; Jain P.; Swamy N.; Bhatia S.; Pradhan H. K. Genetic typing of bovine viral diarrhoea virus isolates from India. Vet. Microbiol. 104: 207–212; 2004.
Mishra N.; Rajukumar K.; Vilcek S.; Tiwari A.; Satav J. S.; Dubey S. C. Molecular characterization of bovine viral diarrhea virus type 2 isolate originating from a native Indian sheep (Ovies aries). Vet. Microbiol. 130: 88–98; 2008.
Nettleton P. F.; Gilray J. A.; Russo P.; Dlissi E. Border disease of sheep and goats. Vet. Res. 29: 327–340; 1998.
Pelkmans L.; Helenius A. Insider information: what viruses tell us about endocytosis. Curr. Opin. Cell Biol. 15: 414–422; 2003.
Pelkmans L.; Kartenbeck J.; Helenius A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat. Cell Biol. 3: 473–483; 2001.
Qu L.; McMullan L. K.; Rice C. M. Isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein NS4B in viral cytopathogenicity. J. Virol. 75: 10651–10662; 2001.
Ronecker S.; Zimmer G.; Herrler G.; Greiser-Wilke I.; Grummer B. Formation of bovine viral diarrhea virus E1–E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains. J. Gen. Virol. 89: 2114–2121; 2008.
Acknowledgments
We thank Dr. Roland Riebe, Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler Institute, Island of Riems, Germany for supplying the SFT-R cell line. This work was supported by a grant from Department of Biotechnology, Govt. of India, New Delhi.
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Editor: J. Denry Sato
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Mathapati, B.S., Mishra, N., Rajukumar, K. et al. Entry of bovine viral diarrhea virus into ovine cells occurs through clathrin-dependent endocytosis and low pH-dependent fusion. In Vitro Cell.Dev.Biol.-Animal 46, 403–407 (2010). https://doi.org/10.1007/s11626-009-9263-9
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DOI: https://doi.org/10.1007/s11626-009-9263-9