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Phenotypic lentivirus screens to identify functional single domain antibodies

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.

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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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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.

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The authors declare no competing financial interests.

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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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