Alberta Institute for Viral Immunology and Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, The University of Alberta, Alberta, Canada.
J Virol. 2010 Mar;84(5):2432-43. doi: 10.1128/JVI.01998-09. Epub 2009 Dec 23.
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.
痘病毒会发生遗传重组，这一点已得到充分证实，但是利用经典遗传杂交来定位痘苗病毒基因的尝试，历史上一直受到实验噪声水平高和物理图谱与遗传图谱之间相关性差的困扰。这些逐个病毒的杂交实验也从未产生过应该在涉及远距离标记的实验中看到的 50%重组子代。痘病毒在称为病毒体（或工厂）的膜包裹细胞质结构中复制，我们开发了一种使用活细胞成像和表达噬菌体 lambda Cro 蛋白融合增强型绿色荧光蛋白（EGFP）的细胞来跟踪这些结构发育的方法。EGFP-cro 蛋白非特异性地结合 DNA，允许对正在发育的痘病毒工厂进行活细胞成像。使用这种方法，我们在感染后约 4 到 5 小时首次看到病毒体。早期病毒体表现出紧凑的外观，然后在持续数小时的指数生长期后，变得模糊并开始消散，这个过程大概与病毒包装有关。在生长期间，病毒体向核周迁移，同时以依赖于感染颗粒数的速率碰撞和融合。然而，即使在高感染复数（10 PFU/细胞）下，我们估计大约 20%的病毒体从不融合。我们还使用荧光原位杂交（FISH）方法研究了由携带不同基因标记的病毒融合形成的病毒体。FISH 显示，融合病毒体内部的 DNA 混合得很差，混合量与病毒体出现和融合之间的时间成反比。我们的研究表明，病毒体的细胞内运动和混合会产生限制，从而减少形成重组体的机会，这些现象产生了反映在经典痘病毒遗传学中的结果。
