Journal of Biological Physics

, Volume 44, Issue 2, pp 181–194 | Cite as

Characterization of AAV vector particle stability at the single-capsid level

  • Julien Bernaud
  • Axel Rossi
  • Anny Fis
  • Lara Gardette
  • Ludovic Aillot
  • Hildegard Büning
  • Martin Castelnovo
  • Anna Salvetti
  • Cendrine Faivre-Moskalenko
Original Paper


Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.


Capsid disassembly Atomic force microscopy Stochastic forces Genome uncoating 



We would like to thank Federico Mingozzi for helpful discussions. This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS; PEPS MPI). It was also funded by grants from the Ecole Normale Supérieure (ENS) de Lyon (to CFM and AS) and Association Française contre les Myopathies (AFM) to AS, HB, and CFM.

Authors’ contributions statement

JB, AR, AS, and CFM conceived and designed the experiments. JB, AF, AR, LG, and AL performed the experiments. JB, AF, AR, MC, HB, AS, and CFM analyzed the data. MC, AS, CFM wrote the paper. .


The authors declare no competing financial interests.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10867_2018_9488_MOESM1_ESM.pdf (5.8 mb)
ESM 1 Supplemental Figure 1 Protocol for the analysis of the AAV response to changes in temperature. Heating of AAV capsids is done for a fixed time at various temperatures ranging from RT to 80 °C before quenching on ice. Mg2+ ions are necessary for DNA binding onto mica, they are added after the heating process as divalent ions are expected to have an effect on capsid stability. The solution is incubated for 5 min on the mica to favor electrostatic adsorption of both DNA and AAV virions. Supplemental Fig. 2: AFM topographic image of AAV8 particles and automated image analysis. (a) raw AFM topographic image of AAV8 capsids, (b) binary image resulting from height and area thresholding, and (c) final topographic image after a few more steps including fractal parameter selection. The further measure of diameter, height or asymmetry on several hundreds of capsids analyzed one by one provides a quantitative morphological characterization of AAV. Supplemental Fig. 3: Automated image analysis of the DNA molecules ejected from the destabilized virions. Using a home-made Matlab script, we can remove the capsids from the image (height thresholding) and skeletonize the DNA fragments in order to measure their length distribution as a function of temperature. Each image with its number corresponds to a different step in the analysis procedure. (a) Initial AFM topographic image. (b) The capsids are removed by applying a height threshold that keep only pixels below 2 nm. (c) Image obtained after the virion has been removed and following by erosion of 1 to 2 pixels around the holes created by capsid removal (height color has been changed). (d) Binary image of DNA only obtained by applying a noise threshold (0.2 nm). (e) The skeleton of DNA filaments can be extracted from the binary image by using morphological tools (erosion). (f) Final topographic image corresponds to image in (c) where the DNA skeleton path has been overlapped in red, as it can clearly been distinguished in the image zoom. Supplemental Fig. 4: AFM imaging of AAV9 virion destabilization induced by heating. Typical AFM images for different heating conditions (a) T = 50 °C, (b) T = 60 °C, (c) T = 65 °C, (d) T = 70 °C, (e) T = 75 °C and (f) T = 80 °C. Each image is shown twice: on the right a color scale of 25 nm is used to see the viral capsids, on the left a 5 nm color scale is chosen to explore what is happening near the surface (ss-DNA width is below 1 nm). At T = 50 °C (a) opened capsids are already observed as DNA is visible on the surface. By increasing the temperature (b), (c) and (d), some filaments can still be observed around the capsids. At higher temperatures (e), both free DNA and DNA linked to capsids exist. At 80 °C mostly free DNA is detected on the surface (f). (PDF 5983 kb)


  1. 1.
    Mingozzi, F., High, K.A.: Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat. Rev. Genet. 12(5), 341–355 (2011). CrossRefGoogle Scholar
  2. 2.
    Asokan, A., Schaffer, D.V., Samulski, R.J.: The AAV vector toolkit: poised at the clinical crossroads. Mol. Ther. : J. Am. Soc. Gene Ther. 20(4), 699–708 (2012). CrossRefGoogle Scholar
  3. 3.
    Buning, H., Huber, A., Zhang, L., Meumann, N., Hacker, U.: Engineering the AAV capsid to optimize vector–host interactions. Curr. Opin. Pharmacol. 24, 94–104 (2015). CrossRefGoogle Scholar
  4. 4.
    Nonnenmacher, M., Weber, T.: Intracellular transport of recombinant adeno-associated virus vectors. Gene Ther. 19(6), 649–658 (2012). CrossRefGoogle Scholar
  5. 5.
    Hauck, B., Zhao, W., High, C., Xiao, W.: Intracellular processing, not single-stranded DNA accumulation, is crucial for recombinant adeno-associated virus transduction. J. Virol. 78, 13678–13686 (2004)CrossRefGoogle Scholar
  6. 6.
    Thomas, C.E., Storm, T.A., Huang, Z., Kay, M.A.: Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. J. Virol. 78(6), 3110–3122 (2004)CrossRefGoogle Scholar
  7. 7.
    Bennett, A., Patel, S., Mietzsch, M., Jose, A., Lins-Austin, B., Yu, J.C., Bothner, B., McKenna, R., Agbandje-McKenna, M.: Thermal stability as a determinant of AAV serotype identity. Mol. Ther. Methods Clin. Dev. 6, 171–182 (2017). CrossRefGoogle Scholar
  8. 8.
    Horowitz, E.D., Rahman, K.S., Bower, B.D., Dismuke, D.J., Falvo, M.R., Griffith, J.D., Harvey, S.C., Asokan, A.: Biophysical and ultrastructural characterization of adeno-associated virus capsid uncoating and genome release. J. Virol. 87(6), 2994–3002 (2013). CrossRefGoogle Scholar
  9. 9.
    Rayaprolu, V., Kruse, S., Kant, R., Venkatakrishnan, B., Movahed, N., Brooke, D., Lins, B., Bennett, A., Potter, T., McKenna, R., Agbandje-McKenna, M., Bothner, B.: Comparative analysis of adeno-associated virus capsid stability and dynamics. J. Virol. 87(24), 13150–13160 (2013). CrossRefGoogle Scholar
  10. 10.
    Venkatakrishnan, B., Yarbrough, J., Domsic, J., Bennett, A., Bothner, B., Kozyreva, O.G., Samulski, R.J., Muzyczka, N., McKenna, R., Agbandje-McKenna, M.: Structure and dynamics of adeno-associated virus serotype 1 VP1-unique N-terminal domain and its role in capsid trafficking. J. Virol. 87(9), 4974–4984 (2013). CrossRefGoogle Scholar
  11. 11.
    Zeng, C., Moller-Tank, S., Asokan, A., Dragnea, B.: Probing the link among genomic cargo, contact mechanics, and Nanoindentation in recombinant adeno-associated virus 2. J. Phys. Chem. B 121(8), 1843–1853 (2017a).
  12. 12.
    Van Kampen, N.G.: Stochastic processes in physics and chemistry, 3rd edition. North-Holland (2007)Google Scholar
  13. 13.
    Salvetti, A., Oreve, S., Chadeuf, G., Favre, D., Cherel, Y., Champion-Arnaud, P., David-Ameline, J., Moullier, P.: Factors influencing recombinant adeno-associated virus production. Hum. Gene Ther. 9(5), 695–706 (1998). CrossRefGoogle Scholar
  14. 14.
    Xiao, X., Li, J., Samulski, R.J.: Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J. Virol. 72(3), 2224–2232 (1998)Google Scholar
  15. 15.
    Wobus, C.E., Hugle-Dorr, B., Girod, A., Petersen, G., Hallek, M., Kleinschmidt, J.A.: Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. J. Virol. 74(19), 9281–9293 (2000)CrossRefGoogle Scholar
  16. 16.
    Faivre-Moskalenko, C., Bernaud, J., Thomas, A., Tartour, K., Beck, Y., Iazykov, M., Danial, J., Lourdin, M., Muriaux, D., Castelnovo, M.: RNA control of HIV-1 particle size polydispersity. PLoS One 9(1), e83874 (2014). ADSCrossRefGoogle Scholar
  17. 17.
    Komura, S., Tamura, K., Kato, T.: Buckling of spherical shells adhering onto a rigid substrate. Eur. Phys. J. E. Soft Matter 18(3), 343–358 (2005). CrossRefGoogle Scholar
  18. 18.
    Zeng, C., Hernando-Perez, M., Dragnea, B., Ma, X., van der Schoot, P., Zandi, R.: Contact mechanics of a small icosahedral virus. Phys. Rev. Lett. 119(3), 038102 (2017b)ADSCrossRefGoogle Scholar
  19. 19.
    Bleker, S., Sonntag, F., Kleinschmidt, J.A.: Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity. J. Virol. 79(4), 2528–2540 (2005). CrossRefGoogle Scholar
  20. 20.
    Kronenberg, S., Bottcher, B., von der Lieth, C.W., Bleker, S., Kleinschmidt, J.A.: A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini. J. Virol. 79(9), 5296–5303 (2005). CrossRefGoogle Scholar
  21. 21.
    Ros, C., Baltzer, C., Mani, B., Kempf, C.: Parvovirus uncoating in vitro reveals a mechanism of DNA release without capsid disassembly and striking differences in encapsidated DNA stability. Virology 345(1), 137–147 (2006). CrossRefGoogle Scholar
  22. 22.
    Luque, A., Zandi, R., Reguera, D.: Optimal architectures of elongated viruses. Proc. Natl. Acad. Sci. U. S. A. 107(12), 5323–5328 (2010). ADSCrossRefGoogle Scholar
  23. 23.
    Reddy, V.S., Giesing, H.A., Morton, R.T., Kumar, A., Post, C.B., Brooks 3rd, C.L., Johnson, J.E.: Energetics of quasiequivalence: computational analysis of protein–protein interactions in icosahedral viruses. Biophys. J. 74(1), 546–558 (1998). ADSCrossRefGoogle Scholar
  24. 24.
    Zandi, R., Reguera, D.: Mechanical properties of viral capsids. Phys. Rev. E Stat. Nonlinear Soft Matter Phys. 72(2 Pt 1), 021917 (2005). MathSciNetCrossRefGoogle Scholar
  25. 25.
    Muthukumar, M.: Polymer escape through a nanopore. J. Chem. Phys. 118, 5174 (2003)ADSCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Julien Bernaud
    • 1
  • Axel Rossi
    • 2
  • Anny Fis
    • 1
  • Lara Gardette
    • 1
  • Ludovic Aillot
    • 2
  • Hildegard Büning
    • 3
  • Martin Castelnovo
    • 1
  • Anna Salvetti
    • 2
  • Cendrine Faivre-Moskalenko
    • 1
  1. 1.Univ Lyon, ENS de LyonUniversité Claude Bernard Lyon 1, CNRS, Laboratoire de PhysiqueLyonFrance
  2. 2.International Center for Infectiology Research (CIRI), Inserm U1111, CNRS UMR5308, Ecole Normale Supérieure de Lyon, LabEx EcofectLyonFrance
  3. 3.Institute of Experimental HematologyHannover Medical SchoolHannoverGermany

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