In previous research, a 27.8-kDa protein in flounder Paralichthys olivaceus 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.IMPORTANCE 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.

中文翻译：

---

电压依赖性阴离子通道蛋白2（VDAC2）和活化蛋白C激酶1（RACK1）的受体充当淋巴囊肿病病毒感染的功能性受体。

在先前的研究中，比目鱼Para鱼（FG）细胞中的27.8 kDa蛋白被鉴定为推定的细胞受体（27.8R），该受体通过与32 kDa病毒附着蛋白相互作用介导了淋巴囊病病毒（LCDV）（开发了LCDV的VAP）和针对27.8R和32 kDa VAP的单克隆抗体（MAb）。在这项研究中，27.8R被确定为电压依赖性阴离子通道蛋白2（VDAC2）和比目鱼活化蛋白C激酶1（RACK1）的受体。通过原核表达获得重组VDAC2（rVDAC2）和RACK1（rRACK1），并制备了兔抗VDAC2 / RACK1多克隆抗体。同时，抗27.8R MAb和抗VDAC2 / RACK1多克隆抗体可识别FG细胞中的rVDAC2，rRACK1和27.8-kDa蛋白。用抗VDAC2 / RACK1多克隆抗体对FG细胞进行预孵育可显着降低LCDV感染细胞的百分比和LCDV拷贝数，阻断病毒感染，并延缓细胞病变作用的发展。LCDV感染后12 h和48 h，FG细胞中VDAC2和RACK1的mRNA表达分别上调至最高水平。与阴性对照相比，通过短干扰RNA（siRNA）进行的VDAC2 / RACK1敲低显着降低了FG细胞中VDAC2 / RACK1的表达和LCDV拷贝数，而LCDV不允许的上皮乳头细胞（EPC）上的VDAC2 / RACK1的表达则更易感染LCDV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充当LCDV进入和感染的受体。重要信息淋巴囊肿病病毒（LCDV）是鱼类淋巴囊肿病的病原体，已给全世界的水产养殖业造成了巨大的经济损失，但分子LCDV与主机交互的基本机制仍不清楚。在这里，LCDV的27.8 kDa推定细胞受体被鉴定为电压依赖性阴离子通道蛋白2（VDAC2）和活化蛋白C激酶1（RACK1）的受体，我们的结果表明VDAC2和RACK1的表达足以使LCDV它们是首次进入LCDV感染的功能性受体，从而使人们更好地了解了LCDV感染和病毒-宿主相互作用的分子机制。重要信息淋巴囊肿病病毒（LCDV）是鱼类淋巴囊肿病的病原体，已经给全世界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，LCDV的27.8 kDa假定细胞受体被鉴定为电压依赖性阴离子通道蛋白2（VDAC2）和活化蛋白C激酶1（RACK1）的受体，我们的结果表明VDAC2和RACK1的表达足以使LCDV它们是首次启动LCDV感染的功能性受体，从而使人们更好地了解了LCDV感染和病毒-宿主相互作用的分子机制。重要信息淋巴囊肿病病毒（LCDV）是鱼类淋巴囊肿病的病原体，已经给全世界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，LCDV的27.8 kDa假定细胞受体被鉴定为电压依赖性阴离子通道蛋白2（VDAC2）和活化蛋白C激酶1（RACK1）的受体，我们的结果表明VDAC2和RACK1的表达足以使LCDV它们是首次启动LCDV感染的功能性受体，从而使人们更好地了解了LCDV感染和病毒-宿主相互作用的分子机制。这给全世界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，LCDV的27.8 kDa假定细胞受体被鉴定为电压依赖性阴离子通道蛋白2（VDAC2）和活化蛋白C激酶1（RACK1）的受体，我们的结果表明VDAC2和RACK1的表达足以使LCDV它们是首次启动LCDV感染的功能性受体，从而使人们更好地了解了LCDV感染和病毒-宿主相互作用的分子机制。这给全世界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，LCDV的27.8 kDa推定细胞受体被鉴定为电压依赖性阴离子通道蛋白2（VDAC2）和活化蛋白C激酶1（RACK1）的受体，我们的结果表明VDAC2和RACK1的表达足以使LCDV它们是首次进入LCDV感染的功能性受体，从而使人们更好地了解了LCDV感染和病毒-宿主相互作用的分子机制。