Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus
Residues 28 to 39 of the Extracellular Loop 1 of Chicken Na+/H+ Exchanger Type I Mediate Cell Binding and Entry of Subgroup J Avian Leukosis Virus
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I型鸡Na/H交换蛋白胞外环1残基28至39介导J亚群禽白血病病毒的细胞结合和进入
DOI：
10.1128/jvi.01627-17
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发表时间：
2018-01-01
影响因子：
5.4
通讯作者：
Gao, Yulong
中科院分区：
文献类型：
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作者：
Guan, Xiaolu;Zhang, Yao;Gao, Yulong
鸡 Na+/H+ 交换蛋白 I 型 (chNHE1) 是一种多跨膜蛋白，是 J 亚组禽白血病病毒 (ALV-J) 的细胞受体。为了鉴定负责 ALV-J 受体活性的 chNHE1 功能决定因素，通过交换人 NHE1 (huNHE1) 和 chNHE1 的细胞外环 (ECL) 以及用血凝素 (HA) 标签替换 ECL，创建了一系列嵌合受体。然后将这些嵌合受体用于结合和进入测定，以绘制 chNHE1 的最小 ALV-J gp85 结合域。我们证明 chNHE1 的 ECL1 (chECL1) 是与 ALV-J gp85 直接相互作用的关键功能 ECL； ECL3 还参与 ALV-J gp85 结合。 chECL1 N 端膜近区的氨基酸残基 28 至 39 构成了 chNHE1 与 ALV-J gp85 结合所需的最小结构域。这些残基足以介导病毒进入 ALV-J 不允许的细胞。点突变分析显示chECL1的A30、V33、W38和E39是介导chNHE1与ALV-J gp85结合的关键残基。此外，用相应的chNHE1残基替换huNHE1的残基28至39将非功能性ALV-J受体huNHE1转变为功能性受体。重要的是，含有 chNHE1 残基 28 至 39 的可溶性 chECL1 和 huECL1 都可以有效阻止 ALV-J 感染。总的来说，我们的研究结果表明，chNHE1 的残基 28 至 39 构成了一个对于受体功能至关重要并介导 ALV-J 进入的结构域。 重要性 chNHE1 是 ALV-J 的细胞受体，ALV-J 是一种逆转录病毒，可导致鸡感染并在家禽业造成严重经济损失。到目前为止，决定 chNHE1 受体功能的结构域仍然未知。我们证明 chECL1 对于受体功能至关重要，残基 28 至 39 构成负责 chNHE1 与 ALV-J gp85 结合并有效介导 ALV-J 细胞进入的最小功能域。这些残基位于chECL1 N末端的近膜区域，表明ALV-J gp85在chNHE1上的结合位点可能位于分子的顶端；受体结合模式可能与逆转录病毒不同。我们还发现可溶性 chECL1 以及含有 chNHE1 残基 28 至 39 的 huECL1 可以有效阻止 ALV-J 感染。这些发现有助于更好地理解ALV-J感染机制，也为ALV-J感染的控制策略提供新的见解。
Chicken Na+/H+ exchanger type I (chNHE1), a multispan transmembrane protein, is a cellular receptor of the subgroup J avian leukosis virus (ALV-J). To identify the functional determinants of chNHE1 responsible for the ALV-J receptor activity, a series of chimeric receptors was created by exchanging the extracellular loops (ECL) of human NHE1 (huNHE1) and chNHE1 and by ECL replacement with a hemagglutinin (HA) tag. These chimeric receptors then were used in binding and entry assays to map the minimal ALV-J gp85-binding domain of chNHE1. We show that ECL1 of chNHE1 (chECL1) is the critical functional ECL that interacts directly with ALV-J gp85; ECL3 is also involved in ALV-J gp85 binding. Amino acid residues 28 to 39 of the N-terminal membrane-proximal region of chECL1 constitute the minimal domain required for chNHE1 binding of ALV-J gp85. These residues are sufficient to mediate viral entry into ALV-J nonpermissive cells. Point mutation analysis revealed that A30, V33, W38, and E39 of chECL1 are the key residues mediating the binding between chNHE1 and ALV-J gp85. Further, the replacement of residues 28 to 39 of huNHE1 with the corresponding chNHE1 residues converted the nonfunctional ALV-J receptor huNHE1 to a functional one. Importantly, soluble chECL1 and huECL1 harboring chNHE1 residues 28 to 39 both could effectively block ALV-J infection. Collectively, our findings indicate that residues 28 to 39 of chNHE1 constitute a domain that is critical for receptor function and mediate ALV-J entry.IMPORTANCE chNHE1 is a cellular receptor of ALV-J, a retrovirus that causes infections in chickens and serious economic losses in the poultry industry. Until now, the domains determining the chNHE1 receptor function remained unknown. We demonstrate that chECL1 is critical for receptor function, with residues 28 to 39 constituting the minimal functional domain responsible for chNHE1 binding of ALV-J gp85 and efficiently mediating ALV-J cell entry. These residues are located in the membrane-proximal region of the N terminus of chECL1, suggesting that the binding site of ALV-J gp85 on chNHE1 is probably located on the apex of the molecule; the receptor-binding mode might be different from that of retroviruses. We also found that soluble chECL1, as well as huECL1 harboring chNHE1 residues 28 to 39, effectively blocked ALV-J infection. These findings contribute to a better understanding of the ALV-J infection mechanism and also provide new insights into the control strategies for ALV-J infection.