Hongyu Cui, Kai Li, Bingling Yun, Yongqiang Wang, Xiaole Qi, Yongzhen Liu, Baoshan Zhang, Mengmeng Yu, Xiaolu Guan, Changjun Liu, Yanni Gao, Yao Zhang, Yulong Gao, Xiaomei Wang, Qing Pan, Yanping Zhang, Chaoqi Ren, Li Gao
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 were then 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--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 non-permissive 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--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--39 could both effectively block ALV-J infection. Collectively, our findings indicate that residues 28--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--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--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.