Clarke Richard W, Monnier Nilah, Li Haitao, Zhou Dejian, Browne Helena, Klenerman David
Department of Chemistry and Division of Virology, University of Cambridge, Cambridge, United Kingdom.
Biophys J. 2007 Aug 15;93(4):1329-37. doi: 10.1529/biophysj.107.106351. Epub 2007 May 18.
We present a single virion method to determine absolute distributions of copy number in the protein composition of viruses and apply it to herpes simplex virus type 1. Using two-color coincidence fluorescence spectroscopy, we determine the virion-to-virion variability in copy numbers of fluorescently labeled tegument and envelope proteins relative to a capsid protein by analyzing fluorescence intensity ratios for ensembles of individual dual-labeled virions and fitting the resulting histogram of ratios. Using EYFP-tagged capsid protein VP26 as a reference for fluorescence intensity, we are able to calculate the mean and also, for the first time to our knowledge, the variation in numbers of gD, VP16, and VP22 tegument. The measurement of the number of glycoprotein D molecules was in good agreement with independent measurements of average numbers of these glycoproteins in bulk virus preparations, validating the method. The accuracy, straightforward data processing, and high throughput of this technique make it widely applicable to the analysis of the molecular composition of large complexes in general, and it is particularly suited to providing insights into virus structure, assembly, and infectivity.
我们提出了一种单病毒粒子方法来确定病毒蛋白质组成中拷贝数的绝对分布，并将其应用于1型单纯疱疹病毒。通过双色符合荧光光谱法，我们通过分析单个双标记病毒粒子集合的荧光强度比并拟合所得的比率直方图，来确定相对于衣壳蛋白的荧光标记被膜蛋白和包膜蛋白拷贝数的病毒粒子间变异性。使用EYFP标记的衣壳蛋白VP26作为荧光强度的参考，我们能够计算平均值，并且据我们所知，首次计算出gD、VP16和VP22被膜蛋白数量的变化。糖蛋白D分子数量的测量结果与大量病毒制剂中这些糖蛋白平均数量的独立测量结果高度一致，验证了该方法。该技术的准确性、直接的数据处理和高通量使其广泛适用于一般大型复合物分子组成的分析，尤其适合于深入了解病毒结构、组装和感染性。
