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Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics

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Human Respiratory Syncytial Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1442))

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Abstract

We present a high-throughput microfluidics platform to identify novel host cell binding partners of respiratory syncytial virus (RSV) matrix (M) protein. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed custom-made gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for binding to RSV M protein.

Even small viral proteome, such as that of RSV, presents a challenge due to the fact that viral proteins are usually multifunctional and thus their interaction with the host is complex. Protein microarrays technology allows the interrogation of protein–protein interactions, which could possibly overcome obstacles by using conventional high throughput methods. Using microfluidics platform we have identified new host interactors of M involved in various cellular pathways. A number of microfluidics based assays have already provided novel insights into the virus–host interactome, and the results have important implications for future antiviral strategies aimed at targets of viral protein interactions with the host.

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References

  1. Gerber D, Maerkl SJ, Quake SR (2009) An in vitro microfluidic approach to generating protein-interaction networks. Nat Methods 6(1):71–74. doi:10.1038/nmeth.1289

    Article  CAS  PubMed  Google Scholar 

  2. Fields S, Song O (1989) A novel genetic system to detect protein-protein interactions. Nature 340(6230):245–246. doi:10.1038/340245a0

    Article  CAS  PubMed  Google Scholar 

  3. Parrish JR, Gulyas KD, Finley RL Jr (2006) Yeast two-hybrid contributions to interactome mapping. Curr Opin Biotechnol 17(4):387–393. doi:10.1016/j.copbio.2006.06.006

    Article  CAS  PubMed  Google Scholar 

  4. Deshaies RJ, Seol JH, McDonald WH, Cope G, Lyapina S, Shevchenko A, Verma R, Yates JR III (2002) Charting the protein complexome in yeast by mass spectrometry. Mol Cell Proteomics 1(1):3–10

    Article  CAS  PubMed  Google Scholar 

  5. Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, Adams SL, Millar A, Taylor P, Bennett K, Boutilier K, Yang L, Wolting C, Donaldson I, Schandorff S, Shewnarane J, Vo M, Taggart J, Goudreault M, Muskat B, Alfarano C, Dewar D, Lin Z, Michalickova K, Willems AR, Sassi H, Nielsen PA, Rasmussen KJ, Andersen JR, Johansen LE, Hansen LH, Jespersen H, Podtelejnikov A, Nielsen E, Crawford J, Poulsen V, Sorensen BD, Matthiesen J, Hendrickson RC, Gleeson F, Pawson T, Moran MF, Durocher D, Mann M, Hogue CW, Figeys D, Tyers M (2002) Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415(6868):180–183. doi:10.1038/415180a

    Article  CAS  PubMed  Google Scholar 

  6. Chen R, Snyder M (2010) Yeast proteomics and protein microarrays. J Proteome 73(11):2147–2157. doi:10.1016/j.jprot.2010.08.003

    Article  CAS  Google Scholar 

  7. Meier M, Sit R, Pan W, Quake SR (2012) High-performance binary protein interaction screening in a microfluidic format. Anal Chem 84(21):9572–9578. doi:10.1021/ac302436y

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Bates SR, Quake SR (2009) Highly parallel measurements of interaction kinetic constants with a microfabricated optomechanical device. Appl Phys Lett 95(7):73705. doi:10.1063/1.3211382

    Article  PubMed  Google Scholar 

  9. Ben-Ari Y, Glick Y, Kipper S, Schwartz N, Avrahami D, Barbiro-Michaely E, Gerber D (2013) Microfluidic large scale integration of viral-host interaction analysis. Lab Chip 13(12):2202–2209. doi:10.1039/c3lc00034f

    Article  CAS  PubMed  Google Scholar 

  10. Glick Y, Avrahami D, Michaely E, Gerber D (2012) High-throughput protein expression generator using a microfluidic platform. J Vis Exp 66, e3849. doi:10.3791/3849

    PubMed  Google Scholar 

  11. Einav S, Gerber D, Bryson PD, Sklan EH, Elazar M, Maerkl SJ, Glenn JS, Quake SR (2008) Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis. Nat Biotechnol 26(9):1019–1027. doi:10.1038/nbt.1490

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Maerkl SJ, Quake SR (2007) A systems approach to measuring the binding energy landscapes of transcription factors. Science 315(5809):233–237. doi:10.1126/science.1131007

    Article  CAS  PubMed  Google Scholar 

  13. Kipper S, Hamad S, Caly L, Avrahami D, Bacharach E, Jans DA, Gerber D, Bajorek M (2015) New host factors important for Respiratory Syncytial Virus replication revealed by a novel microfluidics screen for interactors of matrix protein. Mol Cell Proteomics. doi:10.1074/mcp.M114.044107

    PubMed  PubMed Central  Google Scholar 

  14. Ghildyal R, Ho A, Wagstaff KM, Dias MM, Barton CL, Jans P, Bardin P, Jans DA (2005) Nuclear import of the respiratory syncytial virus matrix protein is mediated by importin beta1 independent of importin alpha. Biochemistry 44(38):12887–12895. doi:10.1021/bi050701e

    Article  CAS  PubMed  Google Scholar 

  15. Ghildyal R, Baulch-Brown C, Mills J, Meanger J (2003) The matrix protein of Human respiratory syncytial virus localises to the nucleus of infected cells and inhibits transcription. Arch Virol 148(7):1419–1429. doi:10.1007/s00705-003-0112-y

    CAS  PubMed  Google Scholar 

  16. Ghildyal R, Mills J, Murray M, Vardaxis N, Meanger J (2002) Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells. J Gen Virol 83(Pt 4):753–757

    Article  CAS  PubMed  Google Scholar 

  17. Ghildyal R, Ho A, Dias M, Soegiyono L, Bardin PG, Tran KC, Teng MN, Jans DA (2009) The respiratory syncytial virus matrix protein possesses a Crm1-mediated nuclear export mechanism. J Virol 83(11):5353–5362. doi:10.1128/JVI.02374-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lifland AW, Jung J, Alonas E, Zurla C, Crowe JE Jr, Santangelo PJ (2012) Human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by MDA5 and MAVS. J Virol 86(15):8245–8258. doi:10.1128/JVI.00215-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li D, Jans DA, Bardin PG, Meanger J, Mills J, Ghildyal R (2008) Association of respiratory syncytial virus M protein with viral nucleocapsids is mediated by the M2-1 protein. J Virol 82(17):8863–8870. doi:10.1128/JVI.00343-08

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bajorek M, Caly L, Tran KC, Maertens GN, Tripp RA, Bacharach E, Teng MN, Ghildyal R, Jans DA (2014) The Thr205 phosphorylation site within respiratory syncytial virus matrix (M) protein modulates M oligomerization and virus production. J Virol. doi:10.1128/JVI.03856-13

    Google Scholar 

  21. Yang X, Boehm JS, Salehi-Ashtiani K, Hao T, Shen Y, Lubonja R, Thomas SR, Alkan O, Bhimdi T, Green TM, Johannessen CM, Silver SJ, Nguyen C, Murray RR, Hieronymus H, Balcha D, Fan C, Lin C, Ghamsari L, Vidal M, Hahn WC, Hill DE, Root DE (2011) A public genome-scale lentiviral expression library of human ORFs. Nat Methods 8(8):659–661. doi:10.1038/nmeth.1638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kusnezow W, Hoheisel JD (2003) Solid supports for microarray immunoassays. J Mol Recognit 16(4):165–176. doi:10.1002/jmr.625

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by European council (ERC #3309600) and Israel Science Foundation (ISF 715/11) (DG), and by European Commission Marie Curie Career Integration Grant (CIG #321931) (MB).

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Authors and Affiliations

  1. Nanotechnology Institute, Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat Gan, Israel

    Sarit Kipper, Dorit Avrahami & Doron Gerber

  2. Section of Virology, Faculty of Medicine, Imperial College London, St. Mary’s Campus, Norfolk Place, London, W2 UK, UK

    Monika Bajorek

Authors
  1. Sarit Kipper
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  2. Dorit Avrahami
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  3. Monika Bajorek
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  4. Doron Gerber
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Corresponding authors

Correspondence to Monika Bajorek or Doron Gerber .

Editor information

Editors and Affiliations

  1. College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

    Ralph A. Tripp

  2. Department of Infectious Diseases, University of Georgia College of Veterinary Medicine, Athens, Georgia, USA

    Patricia A. Jorquera

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Kipper, S., Avrahami, D., Bajorek, M., Gerber, D. (2016). Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics. In: Tripp, R., Jorquera, P. (eds) Human Respiratory Syncytial Virus. Methods in Molecular Biology, vol 1442. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3687-8_12

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  • DOI: https://doi.org/10.1007/978-1-4939-3687-8_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3685-4

  • Online ISBN: 978-1-4939-3687-8

  • eBook Packages: Springer Protocols

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