Visualization of retrovirus budding with correlated light and electron microscopy
Visualization of retrovirus budding with correlated light and electron microscopy
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DOI：
10.1073/pnas.0504812102
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发表时间：
2005-10-25
影响因子：
11.1
通讯作者：
Volker, VM
中科院分区：
文献类型：
--
作者：
Larson, DR;Johnson, MC;Volker, VM
我们已经使用相关扫描电镜（SEM）和多光子荧光显微镜可视化的病毒样颗粒（VLP）的劳斯肉瘤病毒（RSV）和HIV 1型（HIV-1）的出芽。当Gag结构蛋白单独表达为GFP融合体时，大多数出芽颗粒出现形态异常，但正常组装可以通过未标记的Gag蛋白的共表达来拯救。活细胞的成像允许在真实的时间内看到出芽，因为背侧细胞表面的荧光点消失了。包含VLP簇的非常明亮的斑点的消失通常以逐步的方式发生。即使在成像时间>1小时后，也只有少数斑点消失，这表明一些可能是不能出芽的复合物。在单个细胞上，我们列举了通过SEM可见的荧光点和出芽结构，并比较了WT Gag蛋白和在出芽的最后一步被晚期结构域突变阻断的Gag蛋白的这些数字。对于突变的HIV-1和RSV蛋白，几乎所有的荧光点都对应于出芽结构。对于WT RSV，细胞的背侧显示出比出芽结构多3倍的荧光点，表明聚合的Gag壳的形成先于膜的凸出。对于WT HIV-1，大多数荧光斑点对应于出芽结构，与该病毒的较慢出芽速率一致。结合这两种类型的显微镜将允许创新的方法来阐明逆转录病毒出芽的机制。
We have used correlated scanning EM (SEM) and multiphoton fluorescence microscopy to visualize budding of virus-like particles (VLPs) of Rous sarcoma virus (RSV) and HIV type 1 (HIV-1). When the Gag structural protein was expressed alone as a GFP fusion, most budding particles appeared morphologically aberrant, but normal assembly could be rescued by coexpression of untagged Gag protein. Imaging of live cells allowed budding to be seen in real time as the disappearance of fluorescent spots from the dorsal cell surface. The disappearance of very bright spots containing clusters of VLPs often occurred in a stepwise fashion. Even after imaging times >1 h, only a minority of the spots disappeared, suggesting that some might be budding-incompetent complexes. On individual cells, we enumerated both the fluorescent puncta and the budding structures visible by SEM and compared these numbers for WT Gag proteins and for Gag proteins that were blocked at the last step in budding by a late domain mutation. For the mutant HIV-1 and RSV proteins, almost all of the fluorescent spots corresponded to budding structures. For WT RSV, the dorsal side of cells showed 3-fold more fluorescent spots than budding structures, suggesting that formation of the polymerized Gag shell precedes bulging out of the membrane. For WT HIV-1, most fluorescent spots corresponded with budding structures, consistent with the slower budding rate of this virus. Combining these two types of microscopy will allow innovative approaches for elucidating the mechanism of retrovirus budding.