Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton.
Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton.
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DOI：
10.1016/j.jmb.2010.01.043
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发表时间：
2010-03-26
影响因子：
5.6
通讯作者：
Homa FL
中科院分区：
文献类型：
--
作者：
Conway JF;Cockrell SK;Copeland AM;Newcomb WW;Brown JC;Homa FL
单纯疱疹病毒1型(HSV-1)UL25蛋白是DNA切割和包装所需的七种病毒蛋白之一。与UL17一起，UL25形成一种被称为C-衣壳特异成分或CCSC的细长分子的一部分。CCSC的5个副本位于含DNA衣壳的每个衣壳顶端。为了研究UL25在衣壳表面折叠时的构象，我们鉴定了UL25特异性单抗识别的序列，并用免疫金电子显微镜定位了衣壳表面的表位。表位被映射到与衣壳结合所需的蛋白质区域相邻的氨基酸99-111(氨基酸1-50)。此外，将绿色荧光蛋白(GFP)融合在UL25的N端的C-衣壳的冷冻-EM重建定位了UL25与GFP之间的接触点。结果证实了UL25蛋白在CCSC密度中的模拟位置是在五角子的远端，UL25的N末端与从五角子上去掉的三链接触。感染早期的免疫荧光实验表明，UL25-GFP存在于位于细胞质内、靠近细胞核的衣壳上。这些结果支持UL25存在于传入衣壳上的观点，UL25的衣壳结合域位于含有DNA的成熟衣壳的表面。
The herpes simplex virus type 1 (HSV-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 or 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.
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