DOI: 10.1126/science.1064103. Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus

Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus

10.1126/science.1064103

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

We describe a method, based on single-molecule imaging, that allows the real-time visualization of the infection pathway of single viruses in living cells, each labeled with only one fluorescent dye molecule. The tracking of single viruses removes ensemble averaging. Diffusion trajectories with high spatial and time resolution show various modes of motion of adeno-associated viruses (AAV) during their infection pathway into living HeLa cells: (i) consecutive virus touching at the cell surface and fast endocytosis; (ii) free and anomalous diffusion of the endosome and the virus in the cytoplasm and the nucleus; and (iii) directed motion by motor proteins in the cytoplasm and in nuclear tubular structures. The real-time visualization of the infection pathway of single AAVs shows a much faster infection than was generally observed so far.

Bibliography

Seisenberger, G., Ried, M. U., EndreÃ, T., BuÌning, H., Hallek, M., & BraÌuchle, C. (2001). Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus. Science, 294(5548), 1929â1932.

Authors 6

Georg Seisenberger (first)
Martin U. Ried (additional)
Thomas Endreß (additional)
Hildegard Büning (additional)
Michael Hallek (additional)
Christoph Bräuchle (additional)

References 39 Referenced 705

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To characterize the motion of AAV outside the cell we analyzed more than 500 individual displacement steps per time window in 80 AAV-Cy5 trajectories with a distribution function for square displacements derived from the Fick's 2nd law of diffusion (10). We observed a linear dependence of mean square displacements with time (〈r 2 〉 = 4 Dt ) indicative of free diffusion (30). The obtained diffusion coefficient of labeled AAV particles was D = 7.5 μm 2 /s. This is in reasonable agreement with the calculated value ( D = 11 μm 2 /s) derived from the Stokes Einstein equation for AAV assuming a spherical particle with a radius of r = 13 nm.
First measurements with CHO K1 cells and CHO K1 heparan sulfate–negative cells (pgsA-745) show a similar behavior indicating that other parts of the cell membrane not the HSPG receptor may be responsible for this effect. Further experiments are in progress to clarify this point.
In order to characterize the penetration efficiency we determined the ratio of the number of penetration events to the overall number of membrane contact events. Of 847 trajectories starting outside the cell 508 did not show any interaction with the cell. In 271 of the trajectories analyzed the virus particle made membrane contact and then rediffused into the solution. Twenty-five of the particles stuck persistently to the cell membrane. Virus entry into the cell was seen in only 43 of the trajectories. This results in a penetration efficiency of only 13%.
10.1128/JVI.72.2.1438-1445.1998
10.1038/4768
10.1038/4758
10.1083/jcb.138.1.131
10.1016/S0074-7696(08)60527-6
10.1146/annurev.biophys.26.1.373
10.1083/jcb.144.4.657
The start of the experiment is defined here as adding the AAV solution to the sample with the cells.
10.1083/jcb.136.3.531
10.1242/jcs.112.20.3463
www.single-virus-tracing.com.
Virus solution of AAV-Cy5 of low concentrations (10 −9 to 10 −11 mol l −1 ) was added to culture medium (Dulbecco's modified Eagle medium) of HeLa cells. This typically resulted in 10 to 1000 viral particles per cell. An area of 20 μm by 20 μm containing a single cell was imaged with an epifluorescent microscope using a 100× objective (Plan-Neofluar NA 1.3 Zeiss) and a high-sensitivity charge-coupled device (CCD) camera system (Pentamax Princeton Instruments).
H. C. Berg Random Walks in Biology (Princeton Univ. Press Princeton NJ ed. 10 1993).
Cy5 molecules can undergo an average of 10 6 photocycles in buffer/agarose gel before photobleaching. In the experiments described here the excitation intensity was carefully adjusted to achieve sufficient brightness for fluorescence detection but also long trajectories before photobleaching. With a detection efficiency of about ε = 1% and a fluorescence-intensity per spot of several hundred counts trajectories of 1 to 10 s could be obtained.
We are in debt to C. David for valuable discussions and careful reading of the manuscript together with A. Hards. Experimental support by A. Zumbusch T. Hellerer S. Grimm N. Huttner and M. Öhm is gratefully acknowledged.

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:47 a.m.)
Deposited	1 year, 7 months ago (Jan. 9, 2024, 5:35 p.m.)
Indexed	4 weeks ago (Aug. 3, 2025, 6:49 p.m.)
Issued	23 years, 9 months ago (Nov. 30, 2001)
Published	23 years, 9 months ago (Nov. 30, 2001)
Published Print	23 years, 9 months ago (Nov. 30, 2001)

Funders 0

None

BibTeX

@article{Seisenberger_2001, title={Real-Time Single-Molecule Imaging of the Infection Pathway of an Adeno-Associated Virus}, volume={294}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.1064103}, DOI={10.1126/science.1064103}, number={5548}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Seisenberger, Georg and Ried, Martin U. and Endreß, Thomas and Büning, Hildegard and Hallek, Michael and Bräuchle, Christoph}, year={2001}, month=nov, pages={1929–1932} }

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