Virologica Sinica

, Volume 31, Issue 6, pp 500–508 | Cite as

A new strategy for full-length Ebola virus glycoprotein expression in E.coli

  • Junjie Zai
  • Yinhua Yi
  • Han Xia
  • Bo Zhang
  • Zhiming Yuan
Research article

Abstract

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and non-human primates with high rates of fatality. Glycoprotein (GP) is the only envelope protein of EBOV, which may play a critical role in virus attachment and entry as well as stimulating host protective immune responses. However, the lack of expression of full-length GP in Escherichia coli hinders the further study of its function in viral pathogenesis. In this study, the vp40 gene was fused to the full-length gp gene and cloned into a prokaryotic expression vector. We showed that the VP40-GP and GP-VP40 fusion proteins could be expressed in E.coli at 16 °C. In addition, it was shown that the position of vp40 in the fusion proteins affected the yields of the fusion proteins, with a higher level of production of the fusion protein when vp40 was upstream of gp compared to when it was downstream. The results provide a strategy for the expression of a large quantity of EBOV full-length GP, which is of importance for further analyzing the relationship between the structure and function of GP and developing an antibody for the treatment of EBOV infection.

Keywords

Ebola virus (EBOV) glycoprotein (GP) E.coli prokaryotic expression 

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References

  1. Ascenzi P, Bocedi A, Heptonstall J, Capobianchi MR, Di Caro A, Mastrangelo E, Bolognesi M, Ippolito G. 2008. Ebolavirus and Marburgvirus: insight the Filoviridae family. Mol Aspects Med, 29: 151–185.CrossRefPubMedGoogle Scholar
  2. Audet J, Wong G, Wang H, Lu G, Gao GF, Kobinger G, Qiu X. 2014. Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus. Sci Rep, 4: 6881.CrossRefPubMedGoogle Scholar
  3. Bengtsson KL, Song H, Stertman L, Liu Y, Flyer DC, Massare MJ, Xu RH, Zhou B, Lu H, Kwilas SA, Hahn TJ, Kpamegan E, Hooper J, Carrion R, Jr., Glenn G, Smith G. 2016. Matrix-M adjuvant enhances antibody, cellular and protective immune responses of a Zaire Ebola/Makona virus glycoprotein (GP) nanoparticle vaccine in mice. Vaccine, 34: 1927–1935.CrossRefPubMedGoogle Scholar
  4. Bhattacharyya S, Hope TJ. 2011. Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum. Virol J, 8: 11.CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bukreyev A, Rollin PE, Tate MK, Yang L, Zaki SR, Shieh WJ, Murphy BR, Collins PL, Sanchez A. 2007. Successful topical respiratory tract immunization of primates against Ebola virus. J Virol, 81: 6379–6388.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Das D, Jacobs F, Feldmann H, Jones SM, Suresh MR. 2007. Differential expression of the Ebola virus GP(1, 2) protein and its fragments in E. coli. Protein Expr Purif, 54: 117–125.CrossRefPubMedGoogle Scholar
  7. Dolnik O, Volchkova V, Garten W, Carbonnelle C, Becker S, Kahnt J, Stroher U, Klenk HD, Volchkov V. 2004. Ectodomain shedding of the glycoprotein GP of Ebola virus. EMBO J, 23: 2175–2184.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Elliott LH, Sanchez A, Holloway BP, Kiley MP, McCormick JB. 1993. Ebola protein analyses for the determination of genetic organization. Arch Virol, 133: 423–436.CrossRefPubMedGoogle Scholar
  9. Falzarano D, Krokhin O, Wahl-Jensen V, Seebach J, Wolf K, Schnittler HJ, Feldmann H. 2006. Structure-function analysis of the soluble glycoprotein, sGP, of Ebola virus. Chembiochem, 7: 1605–1611.CrossRefPubMedGoogle Scholar
  10. Favier AL, Gout E, Reynard O, Ferraris O, Kleman JP, Volchkov V, Peyrefitte C, Thielens NM. 2016. Enhancement of Ebola virus infection via ficolin-1 interaction with the mucin domain of GP glycoprotein. J Virol, 90: 5256–5269.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Feldmann H, Jones S, Klenk HD, Schnittler HJ. 2003. Ebola virus: from discovery to vaccine. Nat Rev Immunol, 3: 677–685.CrossRefPubMedGoogle Scholar
  12. Hartlieb B, Weissenhorn W. 2006. Filovirus assembly and budding. Virology, 344: 64–70.CrossRefPubMedGoogle Scholar
  13. Jasenosky LD, Neumann G, Lukashevich I, Kawaoka Y. 2001. Ebola virus VP40-induced particle formation and association with the lipid bilayer. J Virol, 75: 5205–5214.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Johnson RF, Bell P, Harty RN. 2006. Effect of Ebola virus proteins GP, NP and VP35 on VP40 VLP morphology. Virol J, 3: 31.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jones SM, Feldmann H, Stroher U, Geisbert JB, Fernando L, Grolla A, Klenk HD, Sullivan NJ, Volchkov VE, Fritz EA, Daddario KM, Hensley LE, Jahrling PB, Geisbert TW. 2005. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat Med, 11: 786–790.CrossRefPubMedGoogle Scholar
  16. Kuhl A, Hoffmann M, Muller MA, Munster VJ, Gnirss K, Kiene M, Tsegaye TS, Behrens G, Herrler G, Feldmann H, Drosten C, Pohlmann S. 2011. Comparative analysis of Ebola virus glycoprotein interactions with human and bat cells. J Infect Dis, 204 Suppl 3: S840–S849.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Lee JE, Fusco ML, Hessell AJ, Oswald WB, Burton DR, Saphire EO. 2008. Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor. Nature, 454: 177–182.CrossRefPubMedPubMedCentralGoogle Scholar
  18. Lee JE, Saphire EO. 2009a. Ebolavirus glycoprotein structure and mechanism of entry. Future Virol, 4: 621–635.CrossRefPubMedPubMedCentralGoogle Scholar
  19. Lee JE, Saphire EO. 2009b. Neutralizing ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it. Curr Opin Struct Biol, 19: 408–417.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Li D, Chen T, Hu Y, Zhou Y, Liu Q, Zhou D, Jin X, Huang Z. 2016. An Ebola Virus-Like Particle-Based Reporter System Enables Evaluation of Antiviral Drugs In Vivo under Non-Biosafety Level 4 Conditions. J Virol, 90: 8720–8728.CrossRefPubMedGoogle Scholar
  21. Licata JM, Johnson RF, Han Z, Harty RN. 2004. Contribution of ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles. J Virol, 78: 7344–7351.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Melito PL, Qiu X, Fernando LM, deVarennes SL, Beniac DR, Booth TF, Jones SM. 2008. The creation of stable cell lines expressing Ebola virus glycoproteins and the matrix protein VP40 and generating Ebola virus-like particles utilizing an ecdysone inducible mammalian expression system. J Virol Methods, 148: 237–243.CrossRefPubMedGoogle Scholar
  23. Mellquist-Riemenschneider JL, Garrison AR, Geisbert JB, Saikh KU, Heidebrink KD, Jahrling PB, Ulrich RG, Schmaljohn CS. 2003. Comparison of the protective efficacy of DNA and baculovirus-derived protein vaccines for EBOLA virus in guinea pigs. Virus Res, 92: 187–193.CrossRefPubMedGoogle Scholar
  24. Noda T, Sagara H, Suzuki E, Takada A, Kida H, Kawaoka Y. 2002. Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP. J Virol, 76: 4855–4865.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Pushko P, Bray M, Ludwig GV, Parker M, Schmaljohn A, Sanchez A, Jahrling PB, Smith JF. 2000. Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus. Vaccine, 19: 142–153.CrossRefPubMedGoogle Scholar
  26. Sanchez A, Kiley MP, Holloway BP, Auperin DD. 1993. Sequence analysis of the Ebola virus genome: organization, genetic elements, and comparison with the genome of Marburg virus. Virus Res, 29: 215–240.CrossRefPubMedGoogle Scholar
  27. Sullivan NJ, Geisbert TW, Geisbert JB, Shedlock DJ, Xu L, Lamoreaux L, Custers JH, Popernack PM, Yang ZY, Pau MG, Roederer M, Koup RA, Goudsmit J, Jahrling PB, Nabel GJ. 2006. Immune protection of nonhuman primates against Ebola virus with single low-dose adenovirus vectors encoding modified GPs. PLoS Med, 3: e177.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Sullivan NJ, Peterson M, Yang ZY, Kong WP, Duckers H, Nabel E, Nabel GJ. 2005. Ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway. J Virol, 79: 547–553.CrossRefPubMedPubMedCentralGoogle Scholar
  29. Sun Y, Carrion R, Jr., Ye L, Wen Z, Ro YT, Brasky K, Ticer AE, Schwegler EE, Patterson JL, Compans RW, Yang C. 2009. Protection against lethal challenge by Ebola virus-like particles produced in insect cells. Virology, 383: 12–21.CrossRefPubMedGoogle Scholar
  30. Swenson DL, Warfield KL, Negley DL, Schmaljohn A, Aman MJ, Bavari S. 2005. Virus-like particles exhibit potential as a panfilovirus vaccine for both Ebola and Marburg viral infections. Vaccine, 23: 3033–3042.CrossRefPubMedGoogle Scholar
  31. Volchkov VE, Feldmann H, Volchkova VA, Klenk HD. 1998. Processing of the Ebola virus glycoprotein by the proprotein convertase furin. Proc Natl Acad Sci U S A, 95: 5762–5767.CrossRefPubMedPubMedCentralGoogle Scholar
  32. Warfield KL, Bosio CM, Welcher BC, Deal EM, Mohamadzadeh M, Schmaljohn A, Aman MJ, Bavari S. 2003. Ebola virus-like particles protect from lethal Ebola virus infection. Proc Natl Acad Sci U S A, 100: 15889–15894.CrossRefPubMedPubMedCentralGoogle Scholar
  33. Warfield KL, Posten NA, Swenson DL, Olinger GG, Esposito D, Gillette WK, Hopkins RF, Costantino J, Panchal RG, Hartley JL, Aman MJ, Bavari S. 2007a. Filovirus-like particles produced in insect cells: Immunogenicity and protection in rodents. Journal of Infectious Diseases, 196: S421–S429.CrossRefPubMedGoogle Scholar
  34. Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, Bavari S. 2007b. Ebola virus-like particle-based vaccine protects nonhuman primates against lethal Ebola virus challenge. J Infect Dis, 196 Suppl 2: S430–S437.CrossRefPubMedGoogle Scholar
  35. Ye L, Lin J, Sun Y, Bennouna S, Lo M, Wu Q, Bu Z, Pulendran B, Compans RW, Yang C. 2006. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies. Virology, 351: 260–270.CrossRefPubMedGoogle Scholar
  36. Zampieri CA, Fortin JF, Nolan GP, Nabel GJ. 2007. The ERK mitogen-activated protein kinase pathway contributes to Ebola virus glycoprotein-induced cytotoxicity. J Virol, 81: 1230–1240.CrossRefPubMedGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2016

Authors and Affiliations

  • Junjie Zai
    • 1
    • 2
  • Yinhua Yi
    • 1
    • 2
  • Han Xia
    • 1
  • Bo Zhang
    • 3
  • Zhiming Yuan
    • 1
  1. 1.Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of VirologyChinese Academy of SciencesWuhanChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases, Wuhan Institute of VirologyChinese Academy of SciencesWuhanChina

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