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

Abstract

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.

Keywords: binding; chicken NHE1; receptors; subgroup J avian leukosis virus; virus entry.

FIG 1

ECL1 and ECL3 participate in the binding of chNHE1 and gp85 protein. (A) Validation of the receptor binding assay. The specific interaction of chNHE1 and gp85-Fc was evaluated using chNHE1-expressing 293T cells by fluorescence-activated cell sorting analysis (FACS). (a) chNHE1-expressing 293T cells incubated with human-Fc protein; (b) chNHE1-expressing 293T cells incubated with FITC-labeled anti-human Fc antibodies; (c) 293T cells expressing nonfunctional huNHE1 receptor incubated with gp85-Fc protein; (d) chNHE1-expressing 293T cells incubated with gp85-Fc protein. (B) Schematic representation of the strategy of constructing chimeric chNHE1 proteins with ECL1 to ECL6 replaced with the corresponding ECL domains of huNHE1 or hemagglutinin (HA) tag. Black, chNHE1 ECLs; white, huNHE1 ELCs; HA, HA tag. (C) The gp85-binding abilities of different chimeric chNHE1s expressed on the surface of transfected 293T cells, evaluated in a receptor binding assay. The binding capacity of wild-type chNHE1 was set to 100%, and the values for chimeric chNHE1 receptors were calculated as its proportion. (D) Entry of RCAS(J)-luciferase virus into 293T cells expressing different chimeric chNHE1s. Virus entry levels were determined using a luciferase reporter assay as described in Materials and Methods. The entry level of RCAS(J)-luciferase virus into 293T cells expressing wild-type chNHE1 was set to 1, and the values for chimeric chNHE1 receptors were calculated as its proportion. (E) Schematic representation of the strategy of constructing chimeric huNHE1s with ECL1 to ECL6 replaced with the corresponding ECLs of chNHE1. The color designations are as described for panel B. (F) The relative gp85-binding capacity of different chimeric huNHE1s expressed on the surface of transfected 293T cells. The binding ability was evaluated in a receptor binding assay. The binding capacity of wild-type chNHE1 was set as 100%, and the values for chimeric chNHE1 receptors were calculated as its proportion. (G) Entry of RCAS(J)-luciferase virus into 293T cells expressing different chimeric huNHE1s, as described for panel D. (B to D) Loss-of-function receptor analysis; (E to G) gain-of-function receptor analysis. (C, D, F, and G) Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P < 0.01.

FIG 2

chECL1 directly interacts with gp85 protein and mediates viral entry. (A) Validation of the interaction of chECL1 and chECL3 with gp85 by a coimmunoprecipitation (co-IP) assay. 293T cells were transfected with gp85- and ECL1- or ECL3-expressing plasmids. HuECL1 and huECL3 were used as a negative control. Cell lysates were prepared 48 h posttransfection, and proteins were immunoprecipitated using anti-HA-agarose MAb; the proteins were immunoblotted with 4A3 anti-gp85 MAb or anti-HA antibodies. (B) Validation of the interaction of chECL1 and chECL3 with gp85 by a pulldown assay. Soluble chECL1 and chECL3 proteins were incubated with anti-HA-agarose MAb. huECL1, huECL3, and human IgG-Fc (huIgG-Fc) were used as a negative control. The bound complexes were mixed with raw lysates of gp85 expressed by 293T cells transfected with pCAF-gp85 plasmid. The proteins were separated by SDS-PAGE and probed by immunoblotting with 4A3 anti-gp85 MAb or anti-HA antibodies. (C) Confocal microscopy analysis of the cell surface expression of ECL1 constructs in 293T cells. 293T cells were transfected with chECL1 and huECL1 expression plasmids for 24 h and fixed and stained with anti-FLAG M2 MAb, followed by incubation with Alexa Fluor 488 donkey anti-mouse IgG(H+L) (green). Nuclei were stained with DAPI. chNHE1 was used as a positive control. (D) The gp85-binding capacity of chECL1 and huECL1 expressed on the surface of transfected 293T cells, evaluated in a receptor binding assay. (E) Entry of RCAS(J)-luciferase virus into 293T cells expressing chECL1 or huECL1, assayed as described in the legend to Fig. 1. (F) Modeling of the chNHE1 transmembrane (TM) domain. For clarity, only TM domains of ECL1 (TM1 and TM2) and ECL3 (TM5 and TM6) are shown. Cell membranes are indicated by dotted lines. The distance between C-terminal portions of TM1 and TM5 is shown to indicate the size of the molecule. The structural modeling of the receptors was generated using SWISS-MODEL and was based on homology molecules found in the PDB database. (D and E) Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment.

FIG 3

Residues 28 to 39 in the N-terminal part of chECL1 constitute the minimal gp85-binding domain. (A) Schematic representation of the strategy for constructing chimeric chNHE1s with three equal-length chECL1 fragments replaced with the corresponding fragments of huECL1. (B) The gp85-binding capacity of 293T cells expressing ECL1-1, ECL1-2, and ECL1-3 chimeric receptors evaluated in a receptor binding assay. (C) Schematic representation of the strategy of constructing chimeric chNHE1s with four equal-length fragments of ECL1-1 replaced with the corresponding fragments of huECL1. (D) The relative gp85-binding capacity of 293T cells expressing ECL1-1A, ECL1-1B, ECL1-1C, and ECL1-1D chimeric receptors, evaluated in a receptor binding assay. (E) Schematic representation of the strategy of constructing chimeric chNHE1s with six equal-length segments of ECL1-1A and ECL1-1B replaced with the corresponding fragments of huECL1. (F) The relative gp85-binding capacity of 293T cells expressing ECL1-1A-1/2/3 and ECL1-1B-1/2/3 chimeric receptors, evaluated in a receptor binding assay. In panels A, C, and E, the black bars represent chECL1 and the white bars represent huECL1. (B, D, and F) Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P < 0.01.

FIG 4

Residues A30, V33, W38, and E39 of ECL1 are key residues for the gp85-binding activity of chNHE1. Eleven amino acids from among residues 28 to 39 of chNHE1 were changed by site-directed mutagenesis to the corresponding amino acids of huNHE1, and the gp85-binding and viral entry-mediating activities of the chimeric receptors were evaluated. (A) Schematic representation of the strategy of constructing chimeric chNHE1s with 11 different single-residue substitutions. (B) The relative gp85-binding capacity of 293T cells expressing the wild type or different single-residue variants of chNHE1. The binding capacity of wild-type chNHE1 was set at 100%, and the values for the other receptors were calculated as proportions of the wild-type value. (C) Entry of RCAS(J)-luciferase virus into 293T cells expressing the wild type or different single-residue variants of chNHE1, assayed as described in the legend to Fig. 1. (B and C) Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P < 0.01.

FIG 5

Residues 28 to 39 of chNHE1 are sufficient to convert huNHE1 into a functional ALV-J receptor and mediate virus entry. (A) Schematic representation of the strategy of constructing chimeric huNHE1s with single-residue or 12-amino-acid substitutions within ECL1. The huNHE1 residues were replaced with the corresponding chNHE1 residues. (B) The relative gp85-binding capacity of 293T cells expressing different chimeric huNHE1s. The binding capacity of wild-type chNHE1 was set at 100%, and the values for the chimeric huNHE1 receptors were calculated as its proportion. (C) Entry of RCAS(J)-luciferase virus into 293T cells expressing different chimeric huNHE1s, assayed as described in the legend to Fig. 1. (B and C) Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P < 0.01.

FIG 6

Soluble huECL1 harboring the corresponding residues 28 to 39 of chNHE1 efficiently blocks ALV-J infection. (A) Coimmunoprecipitation (co-IP) validation of the interaction of gp85 and soluble chimeric huECL1s described in the legend to Fig. 5A. 293T cells were transfected with gp85- and the appropriate soluble chimeric huECL1-expressing plasmids. Cell lysates were prepared 48 h posttransfection, and the proteins were immunoprecipitated by anti-HA-agarose MAb; the proteins were immunoblotted with 4A3 anti-gp85 MAb or anti-HA antibodies. (B) Soluble chECL1 blocks RCAS(J)-luciferase virus entry into DF-1 cells. RCAS(J)-luciferase virus was incubated with different concentrations of soluble chECL1 or bovine serum albumin (BSA) for 1 h at 4°C. This was followed by infection of DF-1 cells. To detect viral entry, the cells infected with RCAS(J)-luciferase virus were lysed 24 h postinfection and luciferase activity was detected in a luciferase reporter assay. BSA was used as a negative control. (C and D) chECL1 and huECL1 harboring the corresponding residues 28 to 39 of chNHE1 block viral entry (C) and reduce the degree of viral replication (D). DF-1 cells were infected with RCAS(J)-luciferase virus or HPRS-103 preincubated with soluble chimeric huECL1s or chECL1. To detect viral entry, the cell culture infected with RCAS(J)-luciferase virus was lysed 24 h postinfection and the luciferase activity was determined by a luciferase reporter assay. To detect viral replication, the cell culture infected with HPRS-103 was harvested 24, 48, and 72 h postinfection, and RT activity was detected using an RT assay. Uninfected DF-1 cells were used as a negative control. RCAS(J)-luciferase virus or HPRS-103 that had not been preincubated with soluble chimeric receptors was used as a positive control. (B, C, and D) Three independent experiments were performed, and data are shown as means ± standard deviations for triplicates from a representative experiment. **, P < 0.01.

FIG 7

Comparison of chNHE1, ECL1, and the receptors of other ALV subgroups and retroviruses. Putative topology of ECL1 and the connected TM domains of chNHE1 are shown. The ALV-J gp85 contour is shown in blue in the size of an HIV-1 gp120 molecule. The identified binding sites on receptors are colored red. The receptors shown are the following: ALV-A, Tva; ALV-J, chNHE1; HIV-1, CD4; ALV-B, -D, and -E, Tvb; ALV-C, Tvc; EIAV, ELR1; and FIV, CD134. CD4 is shown in complex with HIV-1 gp120 (PDB entry 1GC1). The structural modeling of the receptors was generated using SWISS-MODEL and was based on homology molecules found in the PDB database.