Pox, Dyes, and Videotape: Making Movies of GFP-Labeled Vaccinia Virus

The large size of poxvirus virions (approx 250 � 350 nm) makes them ideal candidates for microscopic studies. Recombinant vaccinia viruses that express a viral envelope-specific, green fluorescent protein (GFP) chimera produce enveloped virions that fluoresce green. This fluorescent labeling allows the live, real-time study of viral egress using a variety of microscopic techniques. The methods presented here describe how to image the movement of intracellular enveloped virions that are labeled with green fluorescent protein using time-lapse laser scanning confocal microscopy. Details are also provided for analyzing the images obtained and converting them into QuickTime movies suitable for presentation.