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Tracking viral genomes in host cells at single-molecule resolution


Wang, I-Hsuan; Suomalainen, Maarit; Andriasyan, Vardan; Kilcher, Samuel; Mercer, Jason; Neef, Anne; Luedtke, Nathan W; Greber, Urs F (2013). Tracking viral genomes in host cells at single-molecule resolution. Cell Host & Microbe, 14(4):468-480.

Abstract

Viral 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.

Abstract

Viral 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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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:570 Life sciences; biology
Uncontrolled Keywords:Fluorescence microscopy; virus entry; virus uncoating; nuclear import; foreign DNA;
Language:English
Date:16 October 2013
Deposited On:28 Oct 2013 16:24
Last Modified:05 Apr 2016 17:04
Publisher:Cell Press (Elsevier)
ISSN:1931-3128
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1016/j.chom.2013.09.004
PubMed ID:24139403

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