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

Seisenberger, Georg; Ried, M.; Endreß, Thomas; Büning, Hildegard; Hallek, Michael; Bräuchle, Christoph

doi:10.1126/science.1064103

Cited by 757 publications

(626 citation statements)

References 30 publications

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“…et al, unpublished data). In extreme examples, such as the adeno-associated virus, attaching more than a few dye molecules to the capsid renders the virus noninfectious 6 . This limit can be even more stringent for the labelling of internal viral components, such as the genome.…”

Section: Virus and Cell Labelingmentioning

confidence: 99%

Virus trafficking – learning from single-virus tracking

2007

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What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell? Single-virus tracking in living cells potentially provides us with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.

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Section: Virus and Cell Labelingmentioning

confidence: 99%

Virus trafficking – learning from single-virus tracking

2007

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“…Single AAV particle real-time tracking experiments have shown that AAV uptake into cells occurs in a very rapid manner (within 100 ms). 63 However, studies in intact epithelia have suggested that endocytosis can be a rate-limited factor in rAAV2 infection, and that UV-irradiation can enhance endocytosis of virus. 44 In a manner similar to ligand binding and activation of cellular receptors, AAV binding to cell surface receptors also triggers intracellular signaling pathways that stimulate receptor endocytosis.…”

Section: Receptor-mediated Endocytosis Of Aavmentioning

confidence: 99%

“…However, real-time imaging of fluorescently labeled AAV has suggested that movement of free virus in the cytoplasm and nucleus may be facilitated by ATP-dependent molecular motors on tubular networks. 63 Although Brownian diffusion was also observed, perinuclear delivery of AAV occurred within seconds once anchored to the cytoskeleton.…”

Section: Endosomal Escape and Nuclear Translocation Of Aavmentioning

confidence: 99%

“…40,59,63 However, nuclear transport of AAV in most of the investigated cell lines appears to be a slow and inefficient process. In most studies, only a small portion of internalized AAV can be found in the nucleus at short time points postinfection.…”

Section: Endosomal Escape and Nuclear Translocation Of Aavmentioning

confidence: 99%

“…With the latest advances in fluorescent microscopy, it has become possible to monitor the intracellular movement of single AAV virions in real time. 63 Studies focusing on AAV capsid structures have improved since the crystal structure of AAV2 was determined. Efforts to obtain capsid surface details for other AAV serotypes are accelerating 42,85 and have complemented genetic analyses of the AAV2 capsid that has revealed an enormous tolerance for large insertions such as GFP.…”

Section: Future Directionsmentioning

confidence: 99%

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Intracellular trafficking of adeno-associated viral vectors

et al. 2005

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Adeno-associated virus (AAV) has attracted considerable interest as a gene therapy vector over the past decade. In all, 85% of the current 2052 PubMed references on AAV (as of December 2004) have been published in the last 10 years. As researchers have moved forward with using this vector system for gene delivery, an increased appreciation for the complexities of AAV biology has emerged. The biology of recombinant AAV (rAAV) transduction has demonstrated considerable diversity in different cell types and target tissues. This review will summarize the current understanding of events that control rAAV transduction following receptor binding and leading to nuclear uptake. These stages are broadly classified as intracellular trafficking and have been found to be a major rate-limiting step in rAAV transduction for many cell types. Advances in understanding this area of rAAV biology will help to improve the efficacy of this vector system for the treatment of inherited and acquired diseases. Gene Therapy (2005) 12, 873-880.

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Scanning Dual-Color Cross-Correlation Analysis for Dynamic Co-Localization Studies of Immobile Molecules

et al. 2002

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Dual-color fluorescence cross-correlation analysis has proven to be a powerful tool to probe interactions of different molecular species in solution and living cells on a single molecule level. Probing the coordinated motion of molecules through the measurement volume, it is a much more selective and data-compressing alternative to co-localization analysis by dual-color imaging, and provides additional access to fast internal dynamics of the co-migrating molecules. However, cellular FCS applications often suffer from extremely low molecular mobility, introducing bleaching artifacts or entirely impeding fluctuation analyses of any kind. Thus, to meet the increasing demand for interaction measurements of nearly stationary molecules, such as receptor-ligand complexes on cell membranes, these limitations of conventional fluorescence correlation and cross-correlation analysis need to be overcome. This can be achieved by combining a piezo-driven stage scanning unit with the confocal FCS setup, minimizing the photodynamic strain imposed on immobile single molecules without compromising the relevant cross-correlation information. Different scanning patterns were chosen and the corresponding auto-and cross-correlation curves recorded for both in vitro and in vivo systems. Expectedly, the shape of the correlation curves depends crucially on the different modes of stage motion. Nevertheless, cross-correlation amplitudes clearly reflect on the presence or absence of linkages between the different molecular species. Marked differences between bound and unbound single molecules could be observed on immobilized proteins in PAA gels and on cell membranes.

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

Cited by 757 publications

References 30 publications

Virus trafficking – learning from single-virus tracking

Virus trafficking – learning from single-virus tracking

Intracellular trafficking of adeno-associated viral vectors

Scanning Dual-Color Cross-Correlation Analysis for Dynamic Co-Localization Studies of Immobile Molecules

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