Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
J Mol Biol. 2010 Mar 26;397(2):575-86. doi: 10.1016/j.jmb.2010.01.043. Epub 2010 Jan 25.
The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.
单纯疱疹病毒 1 型 UL25 蛋白是七种病毒蛋白之一，这些蛋白对于 DNA 切割和包装是必需的。UL17 和 UL25 一起形成了一个长分子的一部分，称为 C 衣壳特异性成分（CCSC）。在含 DNA 的衣壳的每个衣壳顶点处，有五个 CCSC 拷贝。为了研究 UL25 在衣壳表面折叠时的构象，我们鉴定了一个 UL25 特异性单克隆抗体识别的序列，并通过免疫金电子显微镜定位了衣壳表面的表位。该表位映射到位于该蛋白（氨基酸 1-50）的衣壳结合所必需的区域的氨基酸 99-111 附近。此外，在将绿色荧光蛋白（GFP）融合到 UL25 的 N 末端的 C 衣壳的 cryo-EM 重建中，定位了 UL25 和 GFP 之间的接触点。结果证实了 UL25 蛋白在 CCSC 密度中的建模位置，即远离五重体的区域，UL25 的 N 末端与远离五重体的三聚体接触。在感染早期的免疫荧光实验表明，UL25-GFP 存在于细胞质中并靠近细胞核的衣壳上。这些结果支持了这样的观点，即 UL25 存在于进入的衣壳上，并且 UL25 的衣壳结合域位于成熟含 DNA 的衣壳的表面上。
