Abstract
Our understanding of infection biology is based on experiments in which pathogen or host proteins are perturbed by small compound inhibitors, mutation, or depletion. This approach has been remarkably successful, as, for example, demonstrated by the independent identification of the endosomal membrane protein Niemann-Pick C1 as an essential factor for Ebola virus infection in both small compound and insertional mutagenesis screens (Côté, Nature 477:344–348, 2011; Carette et al., Nature 477:340–343, 2011). However, many aspects of host-pathogen interactions are poorly understood because we cannot target all of the involved molecules with small molecules, or because we cannot deplete essential proteins. Single domain antibody fragments expressed in the cytosol or other organelles constitute a versatile alternative to perturb the function of any given protein by masking protein-protein interaction interfaces, by stabilizing distinct conformations, or by directly interfering with enzymatic activities. The variable domains of heavy chain-only antibodies (VHHs) from camelid species can be cloned from blood samples of animals immunized with the desired target molecules. We can thus exploit the ability of the camelid immune system to generate affinity-matured single domain antibody fragments to obtain highly specific tools. Interesting VHH candidates are typically identified based on their affinity toward immobilized antigens using techniques such as phage display.
The phenotypical screening approach described here allows the direct identification of VHHs that prevent infection of cells with influenza A virus (IAV) or other pathogens. The VHH repertoire is cloned into a lentiviral vector, which is used to generate pseudo-typed lentivirus particles. Target cells are transduced with the lentivirus, so that every cell inducibly expresses a different VHH. This cell collection is then challenged with a lethal dose of virus. Only the cells which express a VHH that prevents infection by targeting virus proteins or host cell components essential for infection will survive. We can thus identify critical target molecules including vulnerable epitopes and conformations, render target molecules accessible to informative perturbation studies, and stabilize intermediates of virus entry for detailed analysis.
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References
Gargano N, Cattaneo A (1997) Rescue of a neutralizing anti-viral antibody fragment from an intracellular polyclonal repertoire expressed in mammalian cells. FEBS Lett 414:537–540
Helma J, Cardoso MC, Muyldermans S, Leonhardt H (2015) Nanobodies and recombinant binders in cell biology. J Cell Biol 209:633–644. https://doi.org/10.1083/jcb.201409074
Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82:775–797. https://doi.org/10.1146/annurev-biochem-063011-092449
Schmidt FI, Lu A, Chen JW et al (2016) A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly. J Exp Med 213:771–790. https://doi.org/10.1084/jem.20151790
Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB (2007) Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods 324:13–25. https://doi.org/10.1016/j.jim.2007.04.008
Sosa BA, Demircioglu FE, Chen JZ et al (2014) How lamina-associated polypeptide 1 (LAP1) activates Torsin. elife 3:e03239. https://doi.org/10.7554/eLife.03239
Ingram JR, Knockenhauer KE, Markus BM et al (2015) Allosteric activation of apicomplexan calcium-dependent protein kinases. Proc Natl Acad Sci U S A 112:E4975–E4984. https://doi.org/10.1073/pnas.1505914112
Pardon E, Laeremans T, Triest S et al (2014) A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9:674–693. https://doi.org/10.1038/nprot.2014.039
Truttmann MC, Wu Q, Stiegeler S et al (2015) HypE-specific nanobodies as tools to modulate HypE-mediated target AMPylation. J Biol Chem 290:9087–9100. https://doi.org/10.1074/jbc.M114.634287
Ashour J, Schmidt FI, Hanke L et al (2015) Intracellular expression of camelid single-domain antibodies specific for influenza virus nucleoprotein uncovers distinct features of its nuclear localization. J Virol 89:2792–2800. https://doi.org/10.1128/JVI.02693-14
Vanlandschoot P, Stortelers C, Beirnaert E et al (2011) Nanobodies(R): new ammunition to battle viruses. Antivir Res 92:389–407. https://doi.org/10.1016/j.antiviral.2011.09.002
Schmidt FI, Hanke L, Morin B et al (2016) Phenotypic lentivirus screens to identify functional single domain antibodies. Nat Microbiol 1:16080. https://doi.org/10.1038/nmicrobiol.2016.80
Meerbrey KL, Hu G, Kessler JD et al (2011) The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc Natl Acad Sci U S A 108:3665–3670. https://doi.org/10.1073/pnas.1019736108
Barrios-Rodiles M, Brown KR, Ozdamar B et al (2005) High-throughput mapping of a dynamic signaling network in mammalian cells. Science 307:1621–1625. https://doi.org/10.1126/science.1105776
Hanke L, Knockenhauer KE, Brewer RC et al (2016) The antiviral mechanism of an influenza a virus nucleoprotein-specific single-domain antibody fragment. MBio 7:e01569-16. https://doi.org/10.1128/mBio.01569-16
Hanke L, Schmidt FI, Knockenhauer KE et al (2017) Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication. EMBO Rep 18:1027–1037. https://doi.org/10.15252/embr.201643764
Lefranc M-P, Giudicelli V, Ginestoux C et al (1999) IMGT, the international ImMunoGeneTics database. Nucleic Acids Res 27:209–212. https://doi.org/10.1093/nar/27.1.209
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Schmidt, F.I. (2018). Phenotypic Lentivirus Screens to Identify Antiviral Single Domain Antibodies. In: Yamauchi, Y. (eds) Influenza Virus. Methods in Molecular Biology, vol 1836. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8678-1_7
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DOI: https://doi.org/10.1007/978-1-4939-8678-1_7
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