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Phenotypic lentivirus screens to identify functional single domain antibodies

Nature Microbiology volume 1, Article number: 16080 (2016) Cite this article

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Abstract

Manipulation of proteins is key in assessing their in vivo function. Although genetic ablation is straightforward, reversible and specific perturbation of protein function remains a challenge. Single domain antibody fragments, such as camelid-derived VHHs, can serve as inhibitors or activators of intracellular protein function, but functional testing of identified VHHs is laborious. To address this challenge, we have developed a lentiviral screening approach to identify VHHs that elicit a phenotype when expressed intracellularly. We identified 19 antiviral VHHs that protect human A549 cells from lethal infection with influenza A virus (IAV) or vesicular stomatitis virus (VSV), respectively. Both negative-sense RNA viruses are vulnerable to VHHs uniquely specific for their respective nucleoproteins. Antiviral VHHs prevented nuclear import of viral ribonucleoproteins or mRNA transcription, respectively, and may provide clues for novel antiviral reagents. In principle, the screening approach described here should be applicable to identify inhibitors of any pathogen or biological pathway.

To identify the proteins essential to a biological pathway, small-molecule inhibitors or activators may be used to manipulate protein function transiently. Alternatively, screens involving mutagenesis, a reduction in levels or complete elimination of gene products are common1,2. As applied to mammalian cells, these methods usually seek to achieve the removal of a protein from its normal biological context. Many proteins are multifunctional, or are components of multi-subunit complexes. Depletion of any single component may cause unexpected phenotypes due to the collapse of entire protein complexes. Small-molecule inhibitors often lack specificity3 and at best can target a fraction of all the proteins of interest. The screening of chemically diverse libraries must be paired with sophisticated methods to identify the molecular targets of any hit identified. Antibodies have been used as intracellular perturbants of protein function after microinjection4 or cytosolic expression of single-chain variable antibody fragments5, but technical challenges have limited their application to few selected cases.

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Figure 1: Lentiviral screening approach.
Figure 2: Overview of antiviral VHH screen hits.
Figure 3: Validation of antiviral VHHs.
Figure 4: Identification of VHH targets.
Figure 5: Anti-IAV VHHs block nuclear import of vRNPs and mRNA transcription.
Figure 6: Anti-VSV VHHs impair mRNA transcription.

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Acknowledgements

The authors thank B. Bierie for help with lentiviral vectors, M. Taipale and G. Karras for help with LUMIER assays, S. Hulsey Stubbs for VSV-neutralizing antibodies, and T. DiCesare for help with illustrations. This work is supported by a National Institutes of Health Pioneer award to H.L.P. and additional funding from Fujifilm/MediVector. F.I.S. was supported by an Advanced Postdoc.Mobility Fellowship from the Swiss National Science Foundation (SNSF).

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  1. Benjamin Morin

    Present address: † Present address: Agenus Inc., Lexington, Massachusetts 02421, USA.,

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, 02142, Massachusetts, USA

    Florian I. Schmidt, Leo Hanke, Rebeccah Brewer & Hidde L. Ploegh

  2. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Benjamin Morin, Vesna Brusic & Sean P.J. Whelan

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA

    Hidde L. Ploegh

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Contributions

F.I.S., L.H., B.M., R.B. and V.B. performed experiments and analysed the data. S.P.J.W. gave critical technical advice. F.I.S. and H.L.P. conceived the study and wrote the manuscript.

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Correspondence to Hidde L. Ploegh.

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Schmidt, F., Hanke, L., Morin, B. et al. Phenotypic lentivirus screens to identify functional single domain antibodies. Nat Microbiol 1, 16080 (2016). https://doi.org/10.1038/nmicrobiol.2016.80

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