N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1

Abstract

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

Fig 1. Analysis of the impact of gp85 on viral replication.

(A) Virus growth kinetics. DF1 cells are infected with the layer strain JL08CH3-1 and prototype strain HPRS103 at an MOI of 0.01, which are harvested and quantified using a TCID₅₀ assay at 1, 2, 3, 4, 5, 6, and 7 dpi. (B) RT assay. DF1 cells are infected with the layer strain JL08CH3-1 or HPRS103 at an MOI of 0.01, which are harvested and quantified using a colorimetric reverse transcriptase assay at 1, 2, 3, 4, 5, 6, and 7 dpi. (C) Western blotting. The recombinant viruses rHPRS/JL3-1 and rHPRS103 are detected with p27 monoclonal antibody 2E5. (D) IFA assay. DF-1 cells are infected with the recombinant viruses rHPRS/JL3-1 and rHPRS103 for 72 h, and then detected with the 4A3 monoclonal antibody and analyzed using a fluorescence microscope. Scale bar: 400 μm. (E) Virus growth kinetics. DF1 cells are infected with 0.01 MOI rHPRS/JL3-1 and rHPRS103, which are harvested and quantified using a TCID₅₀ assay at 1, 2, 3, 4, 5, 6, and 7 dpi. (F) RT assay. DF1 cells are infected with 0.01 MOI rHPRS/JL3-1 or rHPRS103, which are harvested and quantified using a colorimetric reverse transcriptase assay at 1, 2, 3, 4, 5, 6, and 7 dpi. dpi, days post-inoculation; IFA, immunofluorescence; MOI, multiplicity of infection; TCID₅₀, 50% infective dose of tissue culture.

Fig 2. Detecting the binding capacity of JL08CH3-1-gp85 and HPRS103-gp85 with the receptor chNHE1.

(A) Binding rate between different concentrations of the JL08CH3-1-gp85 and HPRS103-gp85 proteins with chNHE1 expressed on the cell membrane of transfected 293T cells. (B-C) Binding capacity between the rHPRS/JL3-1 and rHPRS103 with chNHE1 expressed on the cell membrane of transfected 293T cells is detected by RT-qPCR (B) and FCAS assays (C). (D-E) The binding capacity is detected by Co-IP assays in 293T cells. (D) 293T cells are co-transfected with chECL1 and pCAF-JL3-1-gp85 or pCAF-gp85 for 48 h. The lysates are incubated with anti-HA-agarose MAb. Lysates are detected by the 4A3 monoclonal antibody and anti-HA monoclonal antibody on Western blotting. (E) The relative intensities of gp85 are normalized to those of gp85 in the input sample. (F) Statistical summary of the KD values of JL08CH3-1-gp85 or HPRS103-gp85 protein and chECL1. (G and H) The binding kinetics of the chECL1 proteins with HPRS103-gp85 (G) or JL08CH3-1-gp85 (H) are detected using Biacore 8K (Cytiva, Marlborough, MA, USA). chECL1 proteins are captured on the chip, and serial dilutions of gp85 then flow through the chip surface. Experiments are performed three times with similar results, and one set of representative data is displayed. Co-IP, coimmunoprecipitation; KD, equilibrium dissociation constant; chECL1, chicken first extracellular loop of chNHE1; chNHE1, chicken sodium hydrogen exchanger isoform 1.

Fig 3. Increased binding of the N123I protein to the receptor.

(A) Amino acid sequence alignment analysis for gp85 from HPRS103 and JL08CH3-1. Green shading represents the chNHE1-binding domain of ALV-J-gp85. The amino acid mutations within the RBD region are marked in red. (B) SDS-PAGE analysis of the JL08CH3-1-gp85, HPRS103-gp85, and 13 mutation gp85 proteins (according to the previous approach to construction). Fifteen plasmids containing CAG-sgp85-Fc, CAG-JL3-1-p85-Fc, and 13 mutation gp85 proteins are transfected into 293T cells for 48 h, then harvested supernatant of cells is purified by using Protein A Resin. Subsequently, the purified gp85 proteins are verified by SDS-PAGE analysis. (C) Binding capacity between the JL08CH3-1-gp85, HPRS103-gp85, and 13 mutation gp85 proteins with chNHE1 expressed on the surface of transfected 293T cells. (D) SDS-PAGE analysis of the N123I-gp85, V128F-gp85, and HPRS103-gp85 proteins. The CAG-sgp85-Fc, CAG-N123I-gp85-Fc, and CAG-V128F-gp85-Fc plasmids are transfected into 293T cells for 48 h, then harvested supernatant of cells is purified using Protein A Resin. Subsequently, the purified gp85 proteins are verified using SDS-PAGE analysis. (E) Binding capacity between the N123I-gp85, V128F-gp85, and HPRS103-gp85 proteins with chNHE1 expressed on the surface of transfected 293T cells. (F and G) The binding capacity is detected by Co-IP assays in 293T cells. (F) 293T cells are co-transfected with chECL1 and pCAF-N123I-gp85 or pCAF-gp85 for 48 h. The lysates are incubated with anti-HA-agarose MAb. Then, the lysates are detected by 4A3 monoclonal antibody and anti-HA monoclonal antibody using Western blotting. (G) Relative intensities of gp85 are normalized to the gp85 in the input sample. (H) Statistical summary of the KD values of N123I-gp85 or HPRS103-gp85 and chECL1. (I and J) The binding kinetics of the chECL1 proteins with HPRS103-gp85(I) or N123I-gp85 (J) are detected using the Biacore 8K (Cytiva, Marlborough, MA, USA). ChECL1 protein is captured on the chip, and serial dilutions of gp85 then flow through the chip surface. Experiments are performed three times, revealing similar results, and one set of representative data is displayed. The error bars represent the SD. *, P < 0.05; **, P < 0.01; *** and ****, P < 0.001; ns, not significant; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RBD, receptor-binding domain; KD, equilibrium dissociation constant; SD, standard deviation; Co-IP, coimmunoprecipitation; chECL1, chicken first extracellular loop of chNHE1; chNHE1, chicken sodium hydrogen exchanger isoform 1.

Fig 4. The modeled structure of N123I-gp85, HPRS103-gp85, and TM-chECL1 proteins, and comparison of molecular docking of both gp85 proteins with TM-chECL1.

(A) The HPRS103-gp85 structure is presented as a ribbon. The side chains of Asn123, Asp125, and Gly206 are displayed. (B) The N123I-gp85 structure is presented as a ribbon. The side chains of Ile123 are displayed. (C) The TM-chECL1 structure is presented as a ribbon. (D) The interaction between HPRS103-gp85 and TM-chECL1 is analyzed using pymol and Ligplot+. (E) The interaction of N123I-gp85 with TM-chECL1 is analyzed by pymol and Ligplot+. The chain on the upside represents gp85; the chain on the downside represents TM-chECL1; the green line represents the hydrogen bond. TM-chECL1, chECL1 with transmembrane region proteins.

Fig 5. Binding free energy calculated for the N123I-gp85, HPRS103-gp85, and TM-chECL1 proteins.

(A) RMSDs of the backbone atoms of HPRS103-gp85-TM-chECL1 complexes. (B) RMSDs of the backbone atoms of N123I-gp85-TM-chECL1 complexes. (C) Binding free energy of HPRS103-gp85 with TM-chECL1. (D) Binding free energy of N123I-gp85 with TM-chECL1. RMSD, root-mean-square deviation; TM-chECL1, chECL1 with transmembrane region proteins.

Fig 6. The replication ability of the recombinant viruses rHPRS103-N123I and rHPRS103 in vivo and in vitro.

(A) The schematic diagram shows the construction of the recombinant virus, rHPRS103-N123I. Using rHPRS103 as the backbone, the Asn123 of rHPRS103 is mutated to Ile123 for constructing the recombinant virus rHPRS103-N123I. (B) Western blotting. The recombinant viruses rHPRS103-N123I and rHPRS103 are detected with the 2E5 monoclonal antibody, rHPRS103 as reference. (C) IFA assay. DF-1 cells are infected with recombinant viruses, rHPRS103-N123I and rHPRS103, for 72 h, and then detected with the 4A3 monoclonal antibody and analyzed using a fluorescence microscope, rHPRS103 as reference. Scale bar: 400 μm. (D and E) Binding capacity between the rHPRS103-N123I and rHPRS103 with chNHE1 expressed on the cell membrane of transfected 293T cells is detected by RT-qPCR (D) and FCAS assays (E). (F) The replication ability of rHPRS103-N123I and rHPRS103 is detected using TCID₅₀ in vitro. DF1 cells are incubated with rHPRS103-N123I and rHPRS103 at an MOI of 0.01, and then harvested and quantified at 1, 2, 3, 4, 5, 6, and 7 dpi. (G) The replication ability of rHPRS103-N123I and rHPRS103 is detected using an RT assay. DF1 cells are incubated with rHPRS103-N123I and rHPRS103 at an MOI of 0.01, and then harvested and quantified 1, 2, 3, 4, 5, 6, and 7 dpi. (H and I) One-day-old SPF chickens were injected intraabdominally with rHPRS103-N123I and rHPRS103 (n = 15, dose = 10⁴ TCID₅₀). (H) Viral loads in whole-blood samples are collected and evaluated using real-time SYBR qPCR mix PCR at different time points. (I) Cloaca swabs are collected and evaluated by ELISA at different time points. The error bars represent the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence; TCID₅₀, 50% tissue culture infective dose; dpi, days post-inoculation; MOI, multiplicity of infection; SPF, specific pathogen-free; qPCR, quantitative polymerase chain reaction; SD, standard deviation.

Fig 7. Phylogenetic tree analysis of the evolutionary relationships between the gp85 gene in various ALV-J strains.

The tree is constructed using the neighbor-joining method with MEGA 6.0 software (https://www.megasoftware.net/home). Bootstrap values are calculated with 1,000 repetitions of the alignment. The three groups are marked. Pink-purple circles represent ALV-J local strains. The blue triangles represent ALV-J layer strains. The red stars represent the ALV-J broiler strains. The N123I mutations within the RBD region of the virus are marked in red. All sequences of ALV-J strains are obtained from GenBank. The accession numbers are listed in the S1 Table.