Characterization of AAV vector particle stability at the single-capsid level
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.
KeywordsCapsid 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.
- 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). https://doi.org/10.1128/JVI.03017-12 CrossRefGoogle Scholar
- 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). https://doi.org/10.1128/JVI.01415-13 CrossRefGoogle Scholar
- 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). https://doi.org/10.1128/JVI.02524-12 CrossRefGoogle Scholar
- 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). https://doi.org/10.1021/acs.jpcb.6b10131
- 12.Van Kampen, N.G.: Stochastic processes in physics and chemistry, 3rd edition. North-Holland (2007)Google Scholar
- 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.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
- 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). https://doi.org/10.1128/JVI.79.4.2528-2540.2005 CrossRefGoogle Scholar
- 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). https://doi.org/10.1128/JVI.79.9.5296-5303.2005 CrossRefGoogle Scholar
- 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). https://doi.org/10.1016/S0006-3495(98)77813-0 ADSCrossRefGoogle Scholar