Grzesik Peter, MacMath Derek, Henson Brandon, Prasad Sanjana, Joshi Poorval, Desai Prashant J
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University, Baltimore, MD, USA.
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University, Baltimore, MD, USA.
Virus Res. 2017 May 15;236:9-13. doi: 10.1016/j.virusres.2017.04.016. Epub 2017 Apr 26.
Self-assembly of herpesvirus capsids can be accomplished in heterologous expression systems provided all six capsid proteins are present. We have demonstrated the assembly of icosahedral Kaposi's sarcoma-associated herpesvirus (KSHV) capsids in insect cells using the baculovirus expression system. Using this self-assembly system we investigated whether we could add additional capsid associated proteins and determine their incorporation into the assembled capsid. We chose the capsid vertex-specific component (CVSC) proteins encoded by open reading frames (ORFs) 19 and 32 to test this. This complex sits on the capsid vertex and is important for capsid maturation in herpesvirus-infected cells. Co-immunoprecipitation assays were used to initially confirm a bi-molecular interaction between ORF19 and ORF32. Both proteins also precipitated the triplex proteins of the capsid shell (ORF26 and ORF62) as well as the major capsid protein (ORF25). Capsid immunoprecipitation assays revealed the incorporation of ORF19 as well as ORF32 into assembled capsids. Similar experiments also showed that the incorporation of each protein occurred independent of the other. These studies reveal biochemically how the KSHV CVSC interacts with the capsid shell.
如果所有六种衣壳蛋白都存在，疱疹病毒衣壳的自组装可以在异源表达系统中完成。我们已经证明，使用杆状病毒表达系统可在昆虫细胞中组装二十面体卡波西肉瘤相关疱疹病毒（KSHV）衣壳。利用这个自组装系统，我们研究了是否可以添加其他衣壳相关蛋白，并确定它们是否能整合到组装好的衣壳中。我们选择了由开放阅读框（ORF）19和32编码的衣壳顶点特异性成分（CVSC）蛋白来进行测试。这种复合物位于衣壳顶点，对疱疹病毒感染细胞中的衣壳成熟很重要。共免疫沉淀试验最初用于证实ORF19和ORF32之间的双分子相互作用。这两种蛋白还沉淀了衣壳壳层的三聚体蛋白（ORF26和ORF62）以及主要衣壳蛋白（ORF25）。衣壳免疫沉淀试验揭示了ORF19和ORF32整合到组装好的衣壳中。类似的实验还表明，每种蛋白的整合都是独立发生的。这些研究从生化角度揭示了KSHV CVSC与衣壳壳层的相互作用方式。
