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Viruses 2018, 10(5), 250; https://doi.org/10.3390/v10050250

Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication

1
Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics Division, Chemistry Department, KU Leuven, B-3001 Leuven, Belgium
2
Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre and Biomedical Research Institute (BIOMED), Hasselt University, B-3590 Diepenbeek, Belgium
*
Author to whom correspondence should be addressed.
Received: 16 March 2018 / Revised: 24 April 2018 / Accepted: 4 May 2018 / Published: 10 May 2018
(This article belongs to the Special Issue Application of Advanced Imaging to the Study of Virus Replication)
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Abstract

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-molecule Förster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resolution microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virology practice. View Full-Text
Keywords: wide-field fluorescence microscopy; confocal laser scanning microscopy; super-resolution microscopy; single virus imaging; raster image correlation spectroscopy; single particle tracking; Förster resonance energy transfer; HIV; simian virus 40, oligomerization; stoichiometry wide-field fluorescence microscopy; confocal laser scanning microscopy; super-resolution microscopy; single virus imaging; raster image correlation spectroscopy; single particle tracking; Förster resonance energy transfer; HIV; simian virus 40, oligomerization; stoichiometry
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Parveen, N.; Borrenberghs, D.; Rocha, S.; Hendrix, J. Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication. Viruses 2018, 10, 250.

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