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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. Access

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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). AUTHOR INFORMATION Author notes * Benjamin Morin Present address: † Present address: Agenus

Inc., Lexington, Massachusetts 02421, USA., AUTHORS AND AFFILIATIONS * Whitehead Institute for Biomedical Research, Cambridge, 02142, Massachusetts, USA Florian I. Schmidt, Leo Hanke, 

Rebeccah Brewer & Hidde L. Ploegh * Department of Microbiology and Immunobiology, Harvard Medical School, Boston, 02115, Massachusetts, USA Benjamin Morin, Vesna Brusic & Sean P.J.

Whelan * Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA Hidde L. Ploegh Authors * Florian I. Schmidt View author publications You can also

search for this author inPubMed Google Scholar * Leo Hanke View author publications You can also search for this author inPubMed Google Scholar * Benjamin Morin View author publications You

can also search for this author inPubMed Google Scholar * Rebeccah Brewer View author publications You can also search for this author inPubMed Google Scholar * Vesna Brusic View author

publications You can also search for this author inPubMed Google Scholar * Sean P.J. Whelan View author publications You can also search for this author inPubMed Google Scholar * Hidde L.

Ploegh View author publications You can also search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Hidde L. Ploegh. ETHICS DECLARATIONS COMPETING

INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures 1-7 (PDF 866 kb) RIGHTS AND PERMISSIONS Reprints

and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 28 January 2016 * Accepted: 28 April 2016 * Published: 20 June 2016 * DOI:

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