Coronaviruses pp 213-229 | Cite as

A Field-Proven Yeast Two-Hybrid Protocol Used to Identify Coronavirus–Host Protein–Protein Interactions

  • Pierre-Olivier Vidalain
  • Yves Jacob
  • Marne C. Hagemeijer
  • Louis M. Jones
  • Grégory Neveu
  • Jean-Pierre Roussarie
  • Peter J. M. Rottier
  • Frédéric Tangy
  • Cornelis A. M. de Haan
Part of the Methods in Molecular Biology book series (MIMB, volume 1282)


Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein–protein interaction networks. In the field of virology, it has proven instrumental to identify virus–host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libraries, and only requires a minimum of equipment. We also discuss reasons that motivated our technical choices and compromises that had to be made. This protocol has been used to screen most non-structural proteins of murine hepatitis virus (MHV), a member of betacoronavirus genus, against a mouse brain cDNA library. Typical results were obtained and are presented in this report.

Key words

Murine hepatitis virus Host–pathogen interactions Yeast two-hybrid Interactomics Proteomics 



This work was supported by Institut Pasteur and CNRS (Centre National de la Recherche Scientifique).


  1. 1.
    Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246CrossRefPubMedGoogle Scholar
  2. 2.
    Uetz P (2012) Editorial for “The Yeast two-hybrid system”. Methods 58:315–316CrossRefPubMedGoogle Scholar
  3. 3.
    Fischer JA, Giniger E, Maniatis T et al (1988) GAL4 activates transcription in Drosophila. Nature 332:853–856CrossRefPubMedGoogle Scholar
  4. 4.
    Keegan L, Gill G, Ptashne M (1986) Separation of DNA binding from the transcription-activating function of a eukaryotic regulatory protein. Science 231:699–704CrossRefPubMedGoogle Scholar
  5. 5.
    Fromont-Racine M, Rain JC, Legrain P (2002) Building protein-protein networks by two-hybrid mating strategy. Methods Enzymol 350:513–524CrossRefPubMedGoogle Scholar
  6. 6.
    Rual JF, Hirozane-Kishikawa T, Hao T et al (2004) Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res 14:2128–2135CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Li S, Armstrong CM, Bertin N et al (2004) A map of the interactome network of the metazoan C. elegans. Science 303:540–543CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Maier RH, Maier CJ, Onder K (2011) Construction of improved yeast two-hybrid libraries. Methods Mol Biol 729:71–84CrossRefPubMedGoogle Scholar
  9. 9.
    Stynen B, Tournu H, Tavernier J et al (2012) Diversity in genetic in vivo methods for protein-protein interaction studies: from the yeast two-hybrid system to the mammalian split-luciferase system. MMBR 76:331–382CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Walhout AJ, Vidal M (2001) High-throughput yeast two-hybrid assays for large-scale protein interaction mapping. Methods 24:297–306CrossRefPubMedGoogle Scholar
  11. 11.
    Mohr K, Koegl M (2012) High-throughput yeast two-hybrid screening of complex cDNA libraries. Methods Mol Biol 812:89–102CrossRefPubMedGoogle Scholar
  12. 12.
    Roberts GG 3rd, Parrish JR, Mangiola BA et al (2012) High-throughput yeast two-hybrid screening. Methods Mol Biol 812:39–61CrossRefPubMedGoogle Scholar
  13. 13.
    Vidalain PO, Boxem M, Ge H et al (2004) Increasing specificity in high-throughput yeast two-hybrid experiments. Methods 32:363–370CrossRefPubMedGoogle Scholar
  14. 14.
    Venkatesan K, Rual JF, Vazquez A et al (2009) An empirical framework for binary interactome mapping. Nat Methods 6:83–90CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Bourai M, Lucas-Hourani M, Gad HH et al (2012) Mapping of Chikungunya virus interactions with host proteins identified nsP2 as a highly connected viral component. J Virol 86:3121–3134CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Braun P, Tasan M, Dreze M et al (2009) An experimentally derived confidence score for binary protein-protein interactions. Nat Methods 6:91–97CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Boxem M, Maliga Z, Klitgord N et al (2008) A protein domain-based interactome network for C. elegans early embryogenesis. Cell 134:534–545CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Caufield JH, Sakhawalkar N, Uetz P (2012) A comparison and optimization of yeast two-hybrid systems. Methods 58:317–324CrossRefPubMedGoogle Scholar
  19. 19.
    Kim MS, Pinto SM, Getnet D et al (2014) A draft map of the human proteome. Nature 509:575–581CrossRefPubMedGoogle Scholar
  20. 20.
    Wilhelm M, Schlegl J, Hahne H et al (2014) Mass-spectrometry-based draft of the human proteome. Nature 509:582–587CrossRefPubMedGoogle Scholar
  21. 21.
    Vidalain PO, Tangy F (2010) Virus-host protein interactions in RNA viruses. Microbes Infect 12:1134–1143CrossRefPubMedGoogle Scholar
  22. 22.
    Friedel CC, Haas J (2011) Virus-host interactomes and global models of virus-infected cells. Trends Microbiol 19:501–508CrossRefPubMedGoogle Scholar
  23. 23.
    Pfefferle S, Schopf J, Kogl M et al (2011) The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7:e1002331CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Millet JK, Kien F, Cheung CY et al (2012) Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage. PLoS One 7:e49566CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Muller M, Cassonnet P, Favre M et al (2013) A comparative approach to characterize the landscape of host-pathogen protein-protein interactions. J Vis Exp. doi: 10.3791/50404 Google Scholar
  26. 26.
    Fielding BC, Gunalan V, Tan TH et al (2006) Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT. Biochem Biophys Res Commun 343:1201–1208CrossRefPubMedGoogle Scholar
  27. 27.
    Titz B, Thomas S, Rajagopala SV et al (2006) Transcriptional activators in yeast. Nucleic Acids Res 34:955–967CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Soellick TR, Uhrig JF (2001) Development of an optimized interaction-mating protocol for large-scale yeast two-hybrid analyses. Genome Biol 2:RESEARCH0052CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Jensen LJ, Kuhn M, Stark M et al (2009) STRING 8—a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 37:D412–D416CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Pierre-Olivier Vidalain
    • 1
    • 2
  • Yves Jacob
    • 3
    • 7
    • 8
  • Marne C. Hagemeijer
    • 5
    • 9
  • Louis M. Jones
    • 6
  • Grégory Neveu
    • 12
    • 13
    • 14
    • 15
  • Jean-Pierre Roussarie
    • 4
    • 10
    • 11
  • Peter J. M. Rottier
    • 4
  • Frédéric Tangy
    • 1
    • 2
  • Cornelis A. M. de Haan
    • 4
  1. 1.Unité de Génomique Virale etVaccinationInstitut PasteurParisFrance
  2. 2.CNRS, UMR3569ParisFrance
  3. 3.Unité de Génétique Moléculaire des Virus à ARN, Département de VirologieInstitut PasteurParisFrance
  4. 4.Laboratory of Molecular and Cellular NeuroscienceThe Rockefeller UniversityNew York USA
  5. 5.Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary MedicineUtrecht UniversityUtrechtThe Netherlands
  6. 6.Centre d’Informatique pour laBiologieInstitut PasteurParisFrance
  7. 7.CNRS, UMR3569ParisFrance
  8. 8.EA302Université Paris Diderot, Sorbonne Paris CitéParisFrance
  9. 9.Laboratory of Host-Pathogen Dynamics, Cell Biology and Physiology Center (CBPC)National Heart, Lung, and Blood Institute (NHLBI), National Institutes of HealthBethesdaUSA
  10. 10.Unité de Génomique Virale etVaccinationInstitut PasteurParisFrance
  11. 11.CNRS, UMR3569ParisFrance
  12. 12.Division of Infectious Diseases and Geographic Medicine, Department of MedicineStanford University School of MedicineStanfordUSA
  13. 13.Unité de Génomique Virale etVaccinationInstitut PasteurParisFrance
  14. 14.CNRS, UMR3569ParisFrance
  15. 15.Department of Microbiology and ImmunologyStanford University School of MedicineStanfordUSA

Personalised recommendations