Yao Zhang , Mengmeng Yu , Lixiao Xing , Peng Liu , Yuntong Chen , Fangfang Chang , Suyan Wang , Yuanling Bao , Muhammad Farooque , Xinyi Li , Xiaolu Guan , Yongzhen Liu , Aijing Liu , Xiaole Qi , Qing Pan , Yanping Zhang , Li Gao , Kai Li , Changjun Liu , Hongyu Cui , Xiaomei Wang , Yulong Gao
J Virol.2020 Sep 2;JVI.01232-20.doi: 10.1128/JVI.01232-20. Online ahead of print.
Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na+/H+ exchanger type I (chNHE1), the 28-39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1-binding in protein-cell binding assays. Our results showed that HA substitutions of amino acids (aa) 38-131 (N-terminal of gp85) and aa 159-283 (C-terminal of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation site and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38-131 and aa 159-283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps.IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of env surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38-131 of the N-terminal and 159-283 of the C-terminal of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C9 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.
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