| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 4361193 | Cell Host & Microbe | 2013 | 13 Pages |
•Tracking incoming single virus DNA genomes by fluorescence microscopy•Cytosolic adenoviruses expose but do not lose their genome before NPC attachment•Cell-cell variable and overall low efficiency of nuclear import of adenovirus DNA•Misdelivery of capsid-free adenovirus DNA to the cytosol upon uncoating at NPC
SummaryViral DNA trafficking in cells has large impacts on physiology and disease development. Current methods lack the resolution and accuracy to visualize and quantify viral DNA trafficking at single-molecule resolution. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresolution microscopy and copper(I)-catalyzed azide-alkyne cycloaddition (click) reactions allowed visualization of infection at single vDNA resolution within mammalian cells. Analysis of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.
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