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Nanomechanical mapping of first binding steps of a virus to animal cells

Abstract

Viral infection is initiated when a virus binds to cell surface receptors. Because the cell membrane is dynamic and heterogeneous, imaging living cells and simultaneously quantifying the first viral binding events is difficult. Here, we show an atomic force and confocal microscopy set-up that allows the surface receptor landscape of cells to be imaged and the virus binding events within the first millisecond of contact with the cell to be mapped at high resolution (<50 nm). We present theoretical approaches to contour the free-energy landscape of early binding events between an engineered virus and cell surface receptors. We find that the first bond formed between the viral glycoprotein and its cognate cell surface receptor has relatively low lifetime and free energy, but this increases as additional bonds form rapidly (≤1 ms). The formation of additional bonds occurs with positive allosteric modulation and the three binding sites of the viral glycoprotein are quickly occupied. Our quantitative approach can be readily applied to study the binding of other viruses to animal cells.

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Figure 1: Principle of combining confocal microscopy and FD-based AFM to image animal cells and to detect virus binding.
Figure 2: Confocal microscopy and FD-based AFM of wild-type MDCK cells and MDCK cells expressing TVA receptors (MDCK–TVA cells) under culturing conditions.
Figure 3: Mapping EnvA–RABV(ΔG) virus binding to MDCK–TVA cells using correlative confocal microscopy and FD-based AFM.
Figure 4: Extracting energetic and kinetic parameters of the EnvA–RABV(ΔG) virus binding to TVA receptors expressed by MDCK cells.
Figure 5: Free-energy landscape describing the binding steps of the EnvA pseudotyped rabies virus to MDCK–TVA cells.

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Acknowledgements

The authors thank T. Lopez and V. Jäggin for assistance with fluorescence-activated cell sorting operation and analysis, M. Mohr for producing eGFP-encoding lentiviruses and E. Bieler, D. Mathys and S. Erpel for assistance with scanning electron microscopy. The plasmid pAAV-EF1a-FLEX-TVA-mCherry was a gift from N. Uchida. The EnvA expressing BHK cell line was a gift from E. Callaway. The Swiss National Science Foundation (SNF; grant no. 310030B_160225 to D.J.M.), NCCR Molecular Systems Engineering and the European Molecular Biology Organization (EMBO; ALTF 265-2013 to D.A. and ALTF 506-2012 to D.M.M.) supported this work. D.A. is Research Associate of FRS-FNRS.

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Contributions

D.A., D.J.M., B.R. and R.N. designed the experiments. R.N. produced viruses, modified cell lines and validated virus-binding and transduction. D.A. and R.S. performed confocal microscopy. D.A. and D.M.-M. set up the AFM chamber. D.A. set up and performed AFM experiments and developed strategies to chemically functionalize the AFM tip. M.D., R.N. and R.S. performed scanning electron microscopy. D.A., D.J.M. and R.N. co-analysed the experimental and calculated data. All authors wrote the paper.

Corresponding authors

Correspondence to David Alsteens or Daniel J. Müller.

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Competing interests

D.A., D.M.M. and D.J.M. have applied for a patent for the chamber enabling AFM and optical microscopy under cell culture conditions (EP15002176.4). The other authors declare no competing financial interests.

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Alsteens, D., Newton, R., Schubert, R. et al. Nanomechanical mapping of first binding steps of a virus to animal cells. Nature Nanotech 12, 177–183 (2017). https://doi.org/10.1038/nnano.2016.228

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