Vo Nguyen T K, Moore Levi C, Leis Eric, DeWitte-Orr Stephanie J
Department of Health Sciences, Wilfrid Laurier University, Waterloo, ON, Canada.
La Crosse Fish Health Center, U.S. Fish and Wildlife Service, Midwest Fisheries Center, Onalaska, WI, USA.
Dev Comp Immunol. 2019 Mar;92:140-149. doi: 10.1016/j.dci.2018.11.012. Epub 2018 Nov 16.
Viral double-stranded (ds)RNA is a potent pathogen-associated molecular pattern (PAMP), capable of inducing a strong antiviral state within the cell, protecting the cell from virus infection. In mammals and fish, sensing extracellular dsRNA is mediated by cell-surface class A scavenger receptors (SR-As). Currently, very little is known about SR-As in amphibians, including: sequence, expression patterns and function. To this end, SR-A expression and function was studied in a novel American toad (Anaxyrus americanus) tadpole cell line called BufoTad. BufoTad was derived from a whole tadpole. The cell line exhibits a cobblestone morphology and expresses abundant levels of transcripts for cytokeratin 19, vimentin, claudin 3, chemokine receptor CXCR4, and SR-AI, one of the five members of the SR-A family, collectively suggesting that BufoTad could be endothelial-like. BufoTad cells bound acetylated LDL, whereas the Xenopus laevis kidney epithelial A6 cell line did not, suggesting functional SR-A activity in BufoTad cells. Additionally, three SR-A competitive ligands (DxSO, fucoidan, poly inosine (pI)) completely blocked AcLDL binding in BufoTad cells, whereas their three corresponding non-competitive ligands (ChSO, fetuin, poly cytosine (pC)) did not. A commercial dsRNA, poly IC, induced robust expression of an Mx-like gene transcript, a possible antiviral protein in BufoTad cells. Employing the same SR-A ligand blocking assay used for AcLDL blocked dsRNA-induced ISG expression. This study is the first demonstration that amphibian SR-As have functional ligand binding activities in a live biological cellular model and that sensing extracellular dsRNA in amphibian cells leads to antiviral gene expression that is mediated by class A scavenger receptors.
病毒双链（ds）RNA是一种有效的病原体相关分子模式（PAMP），能够在细胞内诱导强烈的抗病毒状态，保护细胞免受病毒感染。在哺乳动物和鱼类中，细胞表面A类清道夫受体（SR-A）介导细胞外dsRNA的感知。目前，关于两栖动物的SR-A知之甚少，包括：序列、表达模式和功能。为此，在一种名为BufoTad的新型美洲蟾蜍（Anaxyrus americanus）蝌蚪细胞系中研究了SR-A的表达和功能。BufoTad源自整个蝌蚪。该细胞系呈现鹅卵石形态，表达丰富水平的细胞角蛋白19、波形蛋白、紧密连接蛋白3、趋化因子受体CXCR4以及SR-A家族五个成员之一的SR-AI的转录本，这共同表明BufoTad可能类似内皮细胞。BufoTad细胞结合乙酰化低密度脂蛋白（LDL），而非洲爪蟾肾上皮A6细胞系则不结合，这表明BufoTad细胞具有功能性SR-A活性。此外，三种SR-A竞争性配体（DxSO、岩藻多糖、聚肌苷酸（pI））完全阻断了BufoTad细胞中乙酰化LDL的结合，而它们的三种相应非竞争性配体（ChSO、胎球蛋白、聚胞嘧啶（pC））则没有。一种商业dsRNA，聚肌胞苷酸（poly IC），在BufoTad细胞中诱导了类似Mx基因转录本的强烈表达，这是一种可能的抗病毒蛋白。采用与乙酰化LDL相同的SR-A配体阻断试验可阻断dsRNA诱导的干扰素刺激基因（ISG）表达。这项研究首次证明两栖动物的SR-A在活的生物细胞模型中具有功能性配体结合活性，并且两栖动物细胞中细胞外dsRNA的感知导致由A类清道夫受体介导的抗病毒基因表达。
