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Residues E53, L55, H59, and G70 of the cellular receptor protein Tva mediate cell binding and entry of the novel subgroup K avian leukosis virus.

ABSTRACT: Subgroup K avian leukosis virus (ALV-K) is a novel subgroup of ALV isolated from Chinese native chickens. As for a retrovirus, the interaction between its envelope protein and cellular receptor is a crucial step in ALV-K infection. Tva, a protein previously determined to be associated with vitamin B₁₂/cobalamin uptake, has been identified as the receptor of ALV-K. However, the molecular mechanism underlying the interaction between Tva and the envelope protein of ALV-K remains unclear. In this study, we identified the C-terminal loop of the LDL-A module of Tva as the minimal functional domain that directly interacts with gp85, the surface component of the ALV-K envelope protein. Further point-mutation analysis revealed that E53, L55, H59, and G70, which are exposed on the surface of Tva and are spatially adjacent, are key residues for the binding of Tva and gp85 and facilitate the entry of ALV-K. Homology modeling analysis indicated that the substitution of these four residues did not significantly impact the Tva structure but impaired the interaction between Tva and gp85 of ALV-K. Importantly, the gene-edited DF-1 cell line with precisely substituted E53, L55, H59, and G70 was completely resistant to ALV-K infection and did not affect vitamin B₁₂/cobalamin uptake. Collectively, these findings not only contribute to a better understanding of the mechanism of ALV-K entry into host cells but also provide an ideal gene-editing target for antiviral study.

SUBMITTER: Li X

PROVIDER: S-EPMC9974445 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Residues E53, L55, H59, and G70 of the cellular receptor protein Tva mediate cell binding and entry of the novel subgroup K avian leukosis virus.

Li Xinyi X Chen Yuntong Y Yu Mengmeng M Wang Suyan S Liu Peng P Meng Lingzhai L Guo Ru R Feng Xiaoyan X Hu Mingxue M He Tana T Qi Xiaole X Li Kai K Gao Li L Zhang Yanping Y Liu Changjun C Cui Hongyu H Wang Xiaomei X Gao Yulong Y

The Journal of biological chemistry 20230128 3

Subgroup K avian leukosis virus (ALV-K) is a novel subgroup of ALV isolated from Chinese native chickens. As for a retrovirus, the interaction between its envelope protein and cellular receptor is a crucial step in ALV-K infection. Tva, a protein previously determined to be associated with vitamin B<sub>12</sub>/cobalamin uptake, has been identified as the receptor of ALV-K. However, the molecular mechanism underlying the interaction between Tva and the envelope protein of ALV-K remains unclear. ...[more]

PMID: 36717079

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Project description:The receptor of the subgroup A avian leukosis virus (ALV-A) in chicken is Tva, which is the homologous protein of human CD320 (huCD320), contains a low-density lipoprotein (LDL-A) module and is involved in the uptake of transcobalamin bound vitamin B12/cobalamin (Cbl). To map the functional determinants of Tva responsible for ALV-A receptor activity, a series of chimeric receptors were created by swapping the LDL-A module fragments between huCD320 and Tva. These chimeric receptors were then used for virus entry and binding assays to map the minimal ALV-A functional domain of Tva. The results showed that Tva residues 49 to 71 constituted the minimal functional domain that directly interacted with the ALV-A gp85 protein to mediate ALV-A entry. Single-residue substitution analysis revealed that L55 and W69, which were spatially adjacent on the surface of the Tva structure, were key residues that mediate ALV-A entry. Structural alignment results indicated that L55 and W69 substitutions did not affect the Tva protein structure but abolished the interaction force between Tva and gp85. Furthermore, substituting the corresponding residues of huCD320 with L55 and W69 of Tva converted huCD320 into a functional receptor of ALV-A. Importantly, soluble huCD320 harboring Tva L55 and W69 blocked ALV-A entry. Finally, we constructed a Tva gene-edited cell line with L55R and W69L substitutions that could fully resist ALV-A entry, while Cbl uptake was not affected. Collectively, our findings suggested that amino acids L55 and W69 of Tva were key for mediating virus entry. IMPORTANCE Retroviruses bind to cellular receptors through their envelope proteins, which is a crucial step in infection. While most retroviruses require two receptors for entry, ALV-A requires only one. Various Tva alleles conferring resistance to ALV-A, including Tvar1 (C40W substitution), Tvar2 (frame-shifting four-nucleotide insertion), Tvar3, Tvar4, Tvar5, and Tvar6 (deletion in the first intron), are known. However, the detailed entry mechanism of ALV-A in chickens remains to be explored. We demonstrated that Tva residues L55 and W69 were key for ALV-A entry and were important for correct interaction with ALV-A gp85. Soluble Tva and huCD320 harboring the Tva residues L55 and W69 effectively blocked ALV-A infection. Additionally, we constructed gene-edited cell lines targeting these two amino acids, which completely restricted ALV-A entry without affecting Cbl uptake. These findings contribute to a better understanding of the infection mechanism of ALV-A and provided novel insights into the prevention and control of ALV-A.

Project description: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.

Project description:Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

Dataset Information

Residues E53, L55, H59, and G70 of the cellular receptor protein Tva mediate cell binding and entry of the novel subgroup K avian leukosis virus.

Publications

Residues E53, L55, H59, and G70 of the cellular receptor protein Tva mediate cell binding and entry of the novel subgroup K avian leukosis virus.

Similar Datasets

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