It is well established that poxviruses are subjected to genetic recombination, but attempts to map vaccinia virus genes using classical genetic crosses were historically confounded by high levels of experimental noise and a poor correlation between physical and genetic map distances. These virus-by-virus crosses also never produced the 50% recombinant progeny that should be seen in experiments involving distant markers. Poxviruses replicate in membrane-wrapped cytoplasmic structures called
virosomes (or factories) and we have developed a method for tracking the development of these structures using live cell imaging and cells expressing phage lambda Cro
protein fused to
enhanced green fluorescent protein (EGFP). The EGFP-cro
protein binds nonspecifically to
DNA and permits live cell imaging of developing vaccinia virus factories. Using this method, we see
virosomes first appearing about 4 to 5 h postinfection. The early
virosomes exhibit a compact appearance and then, after a period of exponential growth lasting several hours, blur and start to dissipate in a process presumably linked to viral packaging. During the growth period, the
virosomes migrate toward the nuclear periphery while colliding and fusing at a rate dependent upon the numbers of infecting particles. However, even at high multiplicities of
infection (10 PFU/cell), we estimate approximately 20% of the
virosomes never fuse. We have also used fluorescence in situ hybridization (FISH) methods to study
virosomes formed by the fusion of viruses carrying different gene markers. FISH showed that
DNA mixes rather poorly within fused
virosomes and the amount of mixing is inversely dependent on the time between
virosome appearance and fusion. Our studies suggest that the intracellular movement and mixing of
virosomes create constraints that reduce opportunities for forming recombinants and that these phenomena create outcomes reflected in classical poxvirus genetics.