Zhong Ying, Fei Chenjie, Tang Xiaoqian, Zhan Wenbin, Sheng Xiuzhen
Laboratory of Pathology and Immunology of Aquatic Animals, KLM, Ocean University of China, 5 Yushan Road, Qingdao 266003, PR China.
Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, no. 1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao, PR China.
J Gen Virol. 2017 Jun;98(6):1477-1488. doi: 10.1099/jgv.0.000805. Epub 2017 Jun 20.
The 27.8 kDa protein in flounder gill (FG) cells was previously proved to be a receptor specific for lymphocystis disease virus (LCDV) entry and infection. In this paper, a 32 kDa viral attachment protein (VAP) of LCDV specifically binding to the 27.8 kDa receptor (27.8R) was found by far-Western blotting coupled with monoclonal antibodies (MAbs) against 27.8R. The 32 kDa protein was confirmed to be encoded by the open reading frame (ORF) 038 gene in LCDV-C, and predicted to contain a putative transmembrane region, multiple N-myristoylation and glycosylation sites and phosphorylation motifs. The expression plasmid of pET-32a-ORF038 was constructed and the recombinant VAP (rVAP) was obtained. Rabbit polyclonal antibodies against the rVAP were prepared and could recognize the rVAP and 32 kDa protein in LCDV. Immunogold electron microscopy showed that the 32 kDa protein was located on the surface of LCDV particles. Immunofluorescence assay demonstrated that the rVAP could bind to the 27.8R on the cell membrane of the FG monolayer and the anti-27.8R MAbs could block the rVAP binding. Pre-incubation of the rVAP with FG cells before LCDV infection, or pre-incubation of LCDV with the antibodies against the rVAP, could significantly decrease the LCDV copy numbers (P<0.05) and delay the emergence of cytopathic effects in FG cells in a dose-dependent manner. These results indicated for the first time that the 32 kDa protein functioned as an attachment protein for the initial attachment and entry of LCDV, and the interaction of the 32 kDa VAP with the 27.8R-initiated LCDV infection.
先前已证明，比目鱼鳃（FG）细胞中的27.8 kDa蛋白是淋巴囊肿病病毒（LCDV）进入和感染的特异性受体。在本文中，通过蛋白质免疫印迹结合抗27.8R单克隆抗体（MAbs），发现了LCDV的一种32 kDa病毒附着蛋白（VAP），其能特异性结合27.8 kDa受体（27.8R）。证实该32 kDa蛋白由LCDV - C中的开放阅读框（ORF）038基因编码，并预测其含有一个假定的跨膜区域、多个N - 肉豆蔻酰化和糖基化位点以及磷酸化基序。构建了pET - 32a - ORF038表达质粒并获得重组VAP（rVAP）。制备了针对rVAP的兔多克隆抗体，其可识别rVAP以及LCDV中的32 kDa蛋白。免疫金电子显微镜显示，32 kDa蛋白位于LCDV颗粒表面。免疫荧光分析表明，rVAP可与FG单层细胞膜上的27.8R结合，且抗27.8R单克隆抗体可阻断rVAP的结合。在LCDV感染前将rVAP与FG细胞预孵育，或在LCDV感染前将LCDV与抗rVAP抗体预孵育，均可显著降低LCDV拷贝数（P<0.05），并以剂量依赖方式延迟FG细胞中细胞病变效应的出现。这些结果首次表明，32 kDa蛋白作为LCDV初始附着和进入的附着蛋白发挥作用，且32 kDa VAP与27.8R的相互作用引发了LCDV感染。
