Vaccinia virus particles mix inefficiently, and in a way that would restrict viral recombination, in coinfected cells.

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.