Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, People's Republic of China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, People's Republic of China
J Virol. 2019 May 29;93(12). doi: 10.1128/JVI.00122-19. Print 2019 Jun 15.
In previous research, a 27.8-kDa protein in flounder gill (FG) cells was identified as a putative cellular receptor (27.8R), which mediated lymphocystis disease virus (LCDV) infection via interaction with a 32-kDa viral attachment protein (VAP) of LCDV, and monoclonal antibodies (MAbs) against 27.8R and 32-kDa VAP were developed. In this study, the 27.8R was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1) of flounder. Recombinant VDAC2 (rVDAC2) and RACK1 (rRACK1) were obtained by prokaryotic expression, and rabbit anti-VDAC2/RACK1 polyclonal antibodies were prepared. The rVDAC2, rRACK1, and 27.8-kDa proteins in FG cells were recognized by anti-27.8R MAbs and anti-VDAC2/RACK1 polyclonal antibodies simultaneously. Preincubation of FG cells with anti-VDAC2/RACK1 polyclonal antibodies significantly decreased the percentages of LCDV-infected cells and LCDV copy numbers, blocked virus infection, and delayed the development of cytopathic effect. The mRNA expressions of VDAC2 and RACK1 in FG cells were upregulated to maximum levels 12 h and 48 h after LCDV infection, respectively. VDAC2/RACK1 knockdown through short interfering RNA (siRNA) significantly reduced VDAC2/RACK1 expression and LCDV copy numbers in FG cells compared with negative controls, while VDAC2/RACK1 expression on LCDV-nonpermissive epithelial papillosum cells (EPCs) conferred susceptibility to LCDV infection, indicating the VDAC2 and RACK1 were sufficient to allow LCDV entry and infection. All these results collectively showed that VDAC2 and RACK1 function as receptors for LCDV entry and infection. Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease in fish, which has caused huge economic losses to the aquaculture industry worldwide, but the molecular mechanism underlying the LCDV-host interaction remains unclear. Here, the 27.8-kDa putative cellular receptor for LCDV was identified as voltage-dependent anion channel protein 2 (VDAC2) and receptor of activated protein C kinase 1 (RACK1), and our results revealed that VDAC2 and RACK1 expression was sufficient to allow LCDV entry and that they are functional receptors that initiate LCDV infection for the first time, which leads to a better understanding of the molecular mechanism underlying LCDV infection and virus-host interactions.
在之前的研究中，我们在牙鲆鳃细胞中鉴定出一种 27.8kDa 的蛋白，它是一种假定的细胞受体（27.8R），可通过与淋巴囊肿病病毒（LCDV）的 32kDa 病毒附着蛋白（VAP）相互作用介导病毒感染，并且针对 27.8R 和 32kDa VAP 开发了单克隆抗体（MAbs）。在这项研究中，27.8R 被鉴定为牙鲆电压依赖性阴离子通道蛋白 2（VDAC2）和蛋白激酶 C 激活物受体 1（RACK1）。通过原核表达获得了重组 VDAC2（rVDAC2）和 RACK1（rRACK1），并制备了兔抗 VDAC2/RACK1 多克隆抗体。FG 细胞中的 rVDAC2、rRACK1 和 27.8kDa 蛋白同时被抗 27.8R MAb 和抗 VDAC2/RACK1 多克隆抗体识别。FG 细胞与抗 VDAC2/RACK1 多克隆抗体预孵育可显著降低 LCDV 感染细胞的百分比和 LCDV 拷贝数，阻断病毒感染，并延迟致病变效应的发展。LCDV 感染后 12 小时和 48 小时，FG 细胞中 VDAC2 和 RACK1 的 mRNA 表达水平分别上调至最高水平。与阴性对照相比，通过短发夹 RNA（siRNA）敲低 VDAC2/RACK1 显著降低了 FG 细胞中 VDAC2/RACK1 的表达和 LCDV 拷贝数，而 VDAC2/RACK1 在不允许 LCDV 感染的上皮乳头状细胞（EPCs）上的表达赋予了对 LCDV 感染的易感性，表明 VDAC2 和 RACK1 足以允许 LCDV 进入和感染。所有这些结果共同表明，VDAC2 和 RACK1 作为 LCDV 进入和感染的受体发挥作用。淋巴囊肿病病毒（LCDV）是鱼类淋巴囊肿病的病原体，它给世界范围内的水产养殖业造成了巨大的经济损失，但 LCDV 与宿主相互作用的分子机制仍不清楚。在这里，我们鉴定出 27.8kDa 的 LCDV 假定的细胞受体为电压依赖性阴离子通道蛋白 2（VDAC2）和蛋白激酶 C 激活物受体 1（RACK1），我们的结果首次揭示了 VDAC2 和 RACK1 的表达足以允许 LCDV 进入，并且它们是启动 LCDV 感染的功能性受体，这使得我们对 LCDV 感染和病毒-宿主相互作用的分子机制有了更好的理解。
