The Bipartite Sequence Motif in the N and C Termini of gp85 of Subgroup J Avian Leukosis Virus Plays a Crucial Role in Receptor Binding and Viral Entry
The Bipartite Sequence Motif in the N and C Termini of gp85 of Subgroup J Avian Leukosis Virus Plays a Crucial Role in Receptor Binding and Viral Entry
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J亚型禽白血病病毒gp85 N和C末端的二分序列基序在受体结合和病毒进入中起着至关重要的作用
DOI：
10.1128/jvi.01232-20
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发表时间：
2020-11-01
影响因子：
5.4
通讯作者：
Gao，Yulong
中科院分区：
文献类型：
--
作者：
Zhang，Yao;Yu，Mengmeng;Gao，Yulong
逆转录病毒感染细胞需要宿主和病毒膜的附着和融合。包膜表面蛋白与细胞受体的特异性吸附是引发感染的关键步骤，也是抗病毒药物筛选的靶点。ALV-J是一种经济上重要的禽类病原体，属于甲型逆转录病毒属，其宿主范围比ALV的其他亚群更广。结果表明，ALV-J gp85 N端38~131位氨基酸和C端159~283位氨基酸控制gp85与chNHE1的结合效率，对病毒感染起关键作用。此外，gp85的糖基化位点(N6和N11)和半胱氨酸(C3和C9)在受体结合和病毒进入中起着至关重要的作用。这些发现可能有助于阐明ALV-J进入宿主细胞的机制，并为ALV-J的控制提供抗病毒靶点。J亚群禽白血病病毒(ALV-J)属于甲型逆转录病毒属，通过特异性识别细胞受体鸡Na+/H+交换器I型(ChNHE1)而通过其包膜表面单位(Gp85)进入细胞，其28~39个N端残基被我们以前的研究描述为最小受体功能区。在这项研究中，为了进一步阐明ALV-J gp85与chNHE1相互作用的确切组织和性质，我们利用一系列带有片段替换的gp85突变体鉴定了ALV-J gp85的chNHE1结合域，并在蛋白质-细胞结合实验中评价了它们对chNHE1结合的影响。结果表明，gp85的38~131位氨基酸和C端的159~283位氨基酸的血凝素替换显著抑制了gp85与chNHE1/chNHE1环1的相互作用。此外，这些HA替换的嵌合gp85蛋白不能有效地阻止ALV-J进入chNHE1表达细胞。此外，对gp85的各种N-连接糖基化位点和半胱氨酸突变体的分析表明，糖基化位点(N6和N11)和半胱氨酸(C3和C9)直接参与受体与gp85的结合，并对ALV-J进入细胞起重要作用。综上所述，我们的发现表明，ALV-J gp85的两部分序列基序是与chNHE1结合所必需的，其两个N-连接的糖基化位点和两个半胱氨酸对其受体结合功能和随后的病毒感染步骤是重要的。逆转录病毒对细胞的重要感染需要宿主和病毒膜的附着和融合。包膜表面蛋白与细胞受体的特异性吸附是引发感染的关键步骤，也是抗病毒药物筛选的靶点。ALV-J是一种经济上重要的禽类病原体，属于甲型逆转录病毒属，其宿主范围比ALV的其他亚群更广。结果表明，ALV-J gp85 N端38~131位氨基酸和C端159~283位氨基酸控制gp85与chNHE1的结合效率，对病毒感染起关键作用。此外，gp85的糖基化位点(N6和N11)和半胱氨酸(C3和C9)在受体结合和病毒进入中起着至关重要的作用。这些发现可能有助于阐明ALV-J进入宿主细胞的机制，并为ALV-J的控制提供抗病毒靶点。
Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (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 to 131 of the N terminus and 159 to 283 of the C terminus 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 (C3 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. ABSTRACT 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 to 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 hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus 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 sites 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 to 131 and aa 159 to 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 envelope (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 to 131 of the N terminus and 159 to 283 of the C terminus 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 (C3 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.