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Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination

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Herpes Simplex Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1144))

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Abstract

HSV glycoproteins play important roles in the viral infectious cycle, particularly viral entry into the cell. Here we describe the protocol for expression, purification, and crystallization of viral glycoproteins based on those developed for the HSV-1 gB and HSV-2 gH/gL ectodomains. These protocols can be used for generating milligram amounts of wild-type (WT) or mutant gB and gH/gL ectodomains or can be adapted to produce purified ectodomains of other HSV glycoproteins for biochemical and structural studies.

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References

  1. Heldwein EE, Krummenacher C (2008) Entry of herpesviruses into mammalian cells. Cell Mol Life Sci 65(11):1653–1668. doi:10.1007/s00018-008-7570-z

    Article  PubMed  CAS  Google Scholar 

  2. Johnson DC, Baines JD (2011) Herpesviruses remodel host membranes for virus egress. Nat Rev Microbiol 9(5):382–394. doi:10.1038/nrmicro2559

    Article  PubMed  CAS  Google Scholar 

  3. Johnson DC, Huber MT (2002) Directed egress of animal viruses promotes cell-to-cell spread. J Virol 76(1):1–8

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  4. Showalter SD, Zweig M, Hampar B (1981) Monoclonal antibodies to herpes simplex virus type 1 proteins, including the immediate-early protein ICP 4. Infect Immun 34:684–692

    PubMed Central  PubMed  CAS  Google Scholar 

  5. Eisenberg RJ, Atanasiu D, Cairns TM, Gallagher JR, Krummenacher C, Cohen GH (2012) Herpes virus fusion and entry: a story with many characters. Viruses 4(5):800–832. doi:10.3390/v4050800

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  6. Krummenacher C, Supekar VM, Whitbeck JC, Lazear E, Connolly SA, Eisenberg RJ, Cohen GH, Wiley DC, Carfi A (2005) Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry. EMBO J 24(23):4144–4153

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  7. Heldwein EE, Lou H, Bender FC, Cohen GH, Eisenberg RJ, Harrison SC (2006) Crystal structure of glycoprotein B from herpes simplex virus 1. Science 313(5784):217–220

    Article  PubMed  CAS  Google Scholar 

  8. Atanasiu D, Saw WT, Cohen GH, Eisenberg RJ (2010) Cascade of events governing cell-cell fusion induced by herpes simplex virus glycoproteins gD, gH/gL, and gB. J Virol 84(23):12292–12299. doi:10.1128/JVI.01700-10

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  9. Atanasiu D, Whitbeck JC, Cairns TM, Reilly B, Cohen GH, Eisenberg RJ (2007) Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion. Proc Natl Acad Sci USA 104(47):18718–18723. doi:10.1073/pnas.0707452104, 0707452104 [pii]

    Article  PubMed Central  PubMed  Google Scholar 

  10. Atanasiu D, Whitbeck JC, de Leon MP, Lou H, Hannah BP, Cohen GH, Eisenberg RJ (2010) Bimolecular complementation defines functional regions of Herpes simplex virus gB that are involved with gH/gL as a necessary step leading to cell fusion. J Virol 84(8):3825–3834. doi:10.1128/JVI.02687-09, JVI.02687-09 [pii]

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  11. Stampfer SD, Heldwein EE (2012) Stuck in the middle: structural insights into the role of the gH/gL heterodimer in herpesvirus entry. Curr Opin Virol. doi:10.1016/j.coviro.2012.10.005

    PubMed Central  PubMed  Google Scholar 

  12. Carfi A, Willis SH, Whitbeck JC, Krummenacher C, Cohen GH, Eisenberg RJ, Wiley DC (2001) Herpes simplex virus glycoprotein D bound to the human receptor HveA. Mol Cell 8(1):169–179

    Article  PubMed  CAS  Google Scholar 

  13. Di Giovine P, Settembre EC, Bhargava AK, Luftig MA, Lou H, Cohen GH, Eisenberg RJ, Krummenacher C, Carfi A (2011) Structure of herpes simplex virus glycoprotein D bound to the human receptor nectin-1. PLoS Pathogens 7(9):e1002277. doi:10.1371/journal.ppat.1002277

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  14. Chowdary TK, Cairns TM, Atanasiu D, Cohen GH, Eisenberg RJ, Heldwein EE (2010) Crystal structure of the conserved herpesvirus fusion regulator complex gH-gL. Nat Struct Mol Biol 17(7):882–888. doi:10.1038/nsmb.1837, nsmb.1837 [pii]

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  15. Cocchi F, Fusco D, Menotti L, Gianni T, Eisenberg RJ, Cohen GH, Campadelli-Fiume G (2004) The soluble ectodomain of herpes simplex virus gD contains a membrane-proximal pro-fusion domain and suffices to mediate virus entry. Proc Natl Acad Sci USA 101(19):7445–7450

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  16. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 23(5):567–575. doi:10.1038/nbt1095

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  17. Invitrogen (2010) Bac-to-Bac® Baculovirus Expression System (Version F)

    Google Scholar 

  18. Stampfer SD, Lou H, Cohen GH, Eisenberg RJ, Heldwein EE (2010) Structural basis of local, pH-dependent conformational changes in glycoprotein B from herpes simplex virus type 1. J Virol 84(24):12924–12933. doi:10.1128/JVI.01750-10

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  19. Vitu E, Sharma S, Stampfer SD, Heldwein EE (2013) Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form. J Mol Biol 425(11):2056–2071. doi:10.1016/j.jmb.2013.03.001

    Article  PubMed  CAS  Google Scholar 

  20. Hannah BP, Cairns TM, Bender FC, Whitbeck JC, Lou H, Eisenberg RJ, Cohen GH (2009) Herpes simplex virus glycoprotein B associates with target membranes via its fusion loops. J Virol 83(13):6825–6836. doi:10.1128/JVI.00301-09, JVI.00301-09 [pii]

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  21. Hutchinson L, Browne H, Wargent V, Davis-Poynter N, Primorac S, Goldsmith K, Minson AC, Johnson DC (1992) A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and surface expression of gH. J Virol 66(4):2240–2250

    PubMed Central  PubMed  CAS  Google Scholar 

  22. Roop C, Hutchinson L, Johnson DC (1993) A mutant herpes simplex virus type 1 unable to express glycoprotein L cannot enter cells, and its particles lack glycoprotein H. J Virol 67(4):2285–2297

    PubMed Central  PubMed  CAS  Google Scholar 

  23. Atanasiu D, Cairns TM, Whitbeck JC, Saw WT, Rao S, Eisenberg RJ, Cohen GH (2013) Regulation of herpes simplex virus gB-induced cell-cell fusion by mutant forms of gH/gL in the absence of gD and cellular receptors. MBio 4(2):e00046–13. doi:10.1128/mBio.00046-13

    Article  PubMed Central  PubMed  Google Scholar 

  24. Bender FC, Whitbeck JC, Ponce de Leon M, Lou H, Eisenberg RJ, Cohen GH (2003) Specific association of glycoprotein B with lipid rafts during herpes simplex virus entry. J Virol 77(17):9542–9552

    Article  PubMed Central  PubMed  CAS  Google Scholar 

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Acknowledgments

We acknowledge the contributions of the laboratory of Roselyn Eisenberg and Gary Cohen towards the development of the initial purification protocols of HSV-1 gB730 and HSV-2 gH803/gL produced using recombinant baculoviruses. We also thank Tirumala K. Chowdary and Sapna Sharma for their work in establishing and optimizing these protocols in our laboratory. Finally, we thank past and present members of the Heldwein lab for helpful advice and discussions.

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Authors and Affiliations

  1. Department of Molecular Biology and Microbiology and Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA, 02111, USA

    Samuel D. Stampfer & Ekaterina E. Heldwein

Authors
  1. Samuel D. Stampfer
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  2. Ekaterina E. Heldwein
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Corresponding author

Correspondence to Ekaterina E. Heldwein .

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Editors and Affiliations

  1. Centre for Virus Research, Westmead Millennium Institute, Westmead, New South Wales, Australia

    Russell J. Diefenbach

  2. Institute of Virology, University of Zürich, Zürich, Switzerland

    Cornel Fraefel

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Stampfer, S.D., Heldwein, E.E. (2014). Expression, Purification, and Crystallization of HSV-1 Glycoproteins for Structure Determination. In: Diefenbach, R., Fraefel, C. (eds) Herpes Simplex Virus. Methods in Molecular Biology, vol 1144. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0428-0_17

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  • DOI: https://doi.org/10.1007/978-1-4939-0428-0_17

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0427-3

  • Online ISBN: 978-1-4939-0428-0

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