Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.
Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly.
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DOI：
10.1038/srep13151
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发表时间：
2015-08-19
影响因子：
4.6
通讯作者：
Leong Hew C
中科院分区：
文献类型：
--
作者：
Wang F;Liu Y;Zhu Y;Ngoc Tran B;Wu J;Leong Hew C
新加坡石斑鱼虹彩病毒 (SGIV) 是核细胞质大 DNA 病毒 (NCLDV) 的成员。本文报道了SGIV的主要结构蛋白ORF75R的功能分析。免疫荧光研究表明该蛋白质在病毒组装位点积累。免疫金标记表明它位于病毒衣壳和 DNA 核心之间。吗啉代敲低 ORF75R 导致核壳厚度减少、DNA 衣壳化失败以及感染性颗粒产量低。比较蛋白质组学进一步鉴定了受 ORF75R 敲低影响的结构蛋白。二维凝胶电泳结合蛋白质组学证明，SGIV感染的细胞裂解物和病毒体中ORF75R在多个位点被磷酸化，但病毒体中的绝大多数ORF75R是去磷酸化的亚型。激酶测定表明 ORF75R 可在体外被 SGIV 结构蛋白 ORF39L 磷酸化。将 ATP 和 Mg2+ 添加到纯化的病毒粒子中，促使结构蛋白广泛磷酸化，并从病毒粒子中释放 ORF75R。这些数据表明 ORF75R 是一种新型支架蛋白，对于病毒组装和 DNA 衣壳化很重要，但其磷酸化有利于病毒粒子的分解。与其他病毒的蛋白质相比，我们发现 ORF75R 与单纯疱疹病毒 VP22 具有共同特征。
Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, and the low yield of infectious particles. Comparative proteomics further identified the structural proteins affected by ORF75R knockdown. Two-dimensional gel electrophoresis combined with proteomics demonstrated that ORF75R was phosphorylated at multiple sites in SGIV-infected cell lysate and virions, but the vast majority of ORF75R in virions was the dephosphorylated isoform. A kinase assay showed that ORF75R could be phosphorylated in vitro by the SGIV structural protein ORF39L. Addition of ATP and Mg2+ into purified virions prompted extensive phosphorylation of structural proteins and release of ORF75R from virions. These data suggest that ORF75R is a novel scaffold protein important for viral assembly and DNA encapsidation, but its phosphorylation facilitates virion disassembly. Compared to proteins from other viruses, we found that ORF75R shares common features with herpes simplex virus VP22.