title: Tracking viral genomes in host cells at single-molecule resolution.
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.
authors.affiliation: Institute of Molecular Life Sciences, University of Zürich, CH-8057 Zurich, Switzerland; Molecular Life Sciences Graduate School, ETH and University of Zürich, CH-8057 Zurich, Switzerland.
authors.forename: I-Hsuan
authors.initials: IH
authors.lastname: Wang
authors.forename: Maarit
authors.initials: M
authors.lastname: Suomalainen
authors.forename: Vardan
authors.initials: V
authors.lastname: Andriasyan
authors.forename: Samuel
authors.initials: S
authors.lastname: Kilcher
authors.forename: Jason
authors.initials: J
authors.lastname: Mercer
authors.forename: Anne
authors.initials: A
authors.lastname: Neef
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Luedtke
authors.forename: Urs F
authors.initials: UF
authors.lastname: Greber
mesh_terms: D000256:Adenoviridae / Q000502:physiology*; D001692:Biological Transport; D003600:Cytosol / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis*; D018139:Simplexvirus / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods; D014616:Vaccinia virus / Q000502:physiology*; D014773:Virology / Q000379:methods*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral