Click Chemistry-Based Labeling of Poxvirus Genomes.

Vaccinia virus packages its dsDNA genome inside its core for protection during the extracellular phases of its life cycle. In the cytoplasm of a newly infected cell the viral genome is released from the core so the viral DNA replication machinery can access it and initiate DNA replication. Vaccinia virus replication sites in the cell cytosol can be detected with conventional DNA staining methods; these, however, do not provide enough specificity to be used for quantitative image analysis or further probing of the replication step. Likewise, the ability to generate recombinant vaccinia viruses with fluorescently tagged proteins has provided insight into many stages of the viral life cycle, but many of the early steps involving the viral genome remain to be elucidated. Nucleotide and nucleoside analogs are traditionally used for probing the cell cycle and investigating other changes in cellular DNA, with the more novel nucleoside analogs providing a better way to label with click chemistry. Here we demonstrate how nucleoside analogs and click chemistry can be used for tracking poxvirus replication in the viral factories, and tracking single viral genomes in infected cells.