Hipp Katharina, Schäfer Benjamin, Kepp Gabi, Jeske Holger
Department of Molecular Biology and Plant Virology, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Pfaffenwaldring 57, D-70550 Stuttgart, Germany.
Viruses. 2016 Jul 8;8(7):190. doi: 10.3390/v8070190.
The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses.
双生病毒的衣壳蛋白（CPs）具有多种功能，可用于包装单链病毒基因组、昆虫传播以及在细胞核与细胞质之间穿梭。非洲木薯花叶病毒（ACMV）的CP在裂殖酵母中表达，并通过SDS凝胶电泳进行纯化。对该蛋白进行胰蛋白酶消化后，质谱覆盖了85%的氨基酸序列，并检测到三个N端磷酸化位点（苏氨酸12、丝氨酸25和62）。细胞提取物的差速离心将CP分为两部分，上清液和沉淀。对上清液进行等密度离心时，大部分CP以游离蛋白的典型密度积累，而沉淀部分的CP显示出与核酸的部分结合。上清液CP的尺寸排阻色谱表明存在高阶复合物。在DNA结合试验中，上清液CP加速了单链DNA在琼脂糖凝胶中的迁移，这是颗粒形成的第一个迹象。相应地，等密度离心时CP将单链DNA转移到病毒颗粒的预期密度。然而，电子显微镜未发现任何双生病毒特有的孪生颗粒。
