Visualizing Poxvirus Replication and Recombination Using Live-Cell Imaging

Abstract

A modernized version of an old saying goes that “If a picture is worth a thousand words, then a video is worth a million.” Although made with reference to “YouTube”, the quotation also has relevance for microbiologists when one considers how modern microscopes can be used to track biological fluorophores for hours without bleaching or phototoxicity. Confocal fluorescence microscopy provides a powerful tool for capturing dynamic processes within a cellular context that are better understood when viewed using time-lapse videos. In our laboratory we have long been interested in the links between poxvirus DNA replication and recombination and, since these are cytoplasmic viruses, such DNA-dependent processes are easily imaged throughout the virus life cycle without interference from signals coming from nuclear DNA. In this chapter we outline methods that can be used to follow the movement and replication of vaccinia virus DNA, and to also detect the products of poxvirus-catalyzed recombination reactions. We describe how to use the bacteriophage lambda DNA-binding protein, cro, as a way of labeling DNA within a cell when it is conjugated to fluorescent proteins. When used in conjunction with other fluorescent reagents, new labeling technologies, and tagged reporter constructs, these approaches can generate visually appealing and highly informative insights into diverse aspects of vaccinia virus biology.