Mass spectrometry peptide analyses of Figure 2 gel slices

A giant virus genome is densely packaged by stable nucleosomes within virions. The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 basepairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than “beads-on-a-string” with genic punctuation represents a new mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome. After silver-staining SDS-PAGE, protein from gel slices was extracted for MS analyses, providing trypsinized peptides matched to Marseillevirus predicted protein sequences and ordered based on number of peptides recovered.病毒粒子（virion）内，巨型病毒基因组由稳定的核小体（nucleosome）紧密包裹。马赛病毒（Marseillevirus）的两种双联组蛋白与真核生物的四类核心组蛋白亲缘关系较远，可包裹121个碱基对的DNA，形成结构极为相似的核小体。通过透化马赛病毒粒子并开展全基因组范围的核酸酶消化、化学裂解及质谱（mass spectrometry）分析实验，我们发现马赛病毒染色质的高级组织结构与真核生物存在根本性差异。马赛病毒的核小体可完全保护病毒粒子内的DNA，形成紧密邻接的121 bp DNA包裹核心结构，不存在连接DNA（linker DNA）或沿基因的相位排布。同理，我们观察到在核小体定位序列的多拷贝串联重复序列上重建的核小体同样呈紧密包裹状态。完全包裹的核小体呈致密且无序的紧密排布，而非带有基因分隔标记的串珠状（beads-on-a-string）结构，这代表了组蛋白介导DNA包装的一种全新模式。我们推测，双联组蛋白的演化是为了保护病毒基因组，其可能对应了组蛋白分化形成真核生物八聚体核小体的早期阶段。经银染的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）后，研究人员从凝胶切片中提取蛋白质用于质谱分析，所得胰酶消化肽段可匹配马赛病毒的预测蛋白质序列，并依据回收肽段的数量进行排序。