Identification Of Receptor-binding Domain Of Subgroup J Avian Leukosis Virus Envelop Protein And Characterization Of Distinction In Binding Affinity

Avian leukosis of subgroup J is a neoplastic and infectious disease induced by subgroup J avian leukosis virus（ALV-J）.The first detection of ALV-J was from commercial broiler.In recent years,ALV-J is prevalent in layer flock and local flock with greater pathogenicity.The host range of ALV-J has enlarged significantly and the envelop protein of ALV-J has been mutated frenquently.Protein gp85is the surface unit protein of ALV-J envelop which contains the virus-receptor determinants.Gp85 was in charge of recognizing the specific receptor and then determine the subgroup specificity and host range.The change of host range of ALV-J was closely related to the evolution of gp85 sequence.In this study,we identified the receptor-binding domain of ALV-J gp85.Moreover the distinction in gp85-chNHE1 binding affinity of different strains of ALV-J induced by the difference of gp85 amino acid sequences was charactered.Previous studies have demonstrated that receptor-binding domain of gp85 of ALV subgroup A to E were identified as hr1 and hr2.To identify the receptor-binding domain of ALV-J gp85,we constructed a chimeric plasmid that could express soluble gp85 of HPRS-103 with biological activity.Then a series segment substitution mutants（33）of ALV-J gp85 by HA tag was constructed.After expression and purification of these 33 gp85 proteins,the binding capacities of these protein to chNHE1was evaluated by of receptor binding assay.The result indicates that 23 gp85 lost the binding capacity with chNHE1.Furthermore,the blocking virus entry assay was confirmed to the binding result that the receptor-binding domain is located on 37 to 130,159 to 167 and 180 to 283 amino acid.By sequence align of ALV-J gp85,we found that there exist many amino acids mutation between gp85 sequence of domestic epidemic strains and gp85 sequence of broiler isolates represented by HPRS-103.The result of replication kinetics assay suggested that the replication ability of domestic epidemic strain JL08CH3-1 was higher than prototype strain HPRS-103.Receptor binding assay suggested that the binding capacity of JL08CH3-1 gp85（JL3-1-gp85）to ch NHE1 was higher than HPRS-103 gp85（HPRS-103-gp85）to chNHE1.The difference in binding capacity of them was increased as the concentration decreased.To investigate whether the difference of replication ability or binding capacity was related to these regular amino acid mutations,we aligned the amino acid sequence of the two groups of gp85 and 23 different amino acid sites were identified.We conducted a group of point mutation on the different amino acids between JL3-1-gp85 and HPRS-103-gp85,based on the sequence of HPRS-103-gp85.By receptor binding assay,the binding capacity of amino acids 114,117and 123 mutation(HPRS-103-gp85 ^{E114-/R117G/N123I})to chNHE1 was increased and close to JL3-1-gp85’s.To further confirm the difference in binding capacity of different gp85 with chNHE1,we expressed the first extracellular loop of chNHE1 named ch ECL1 which was supposed to be the functional domain of chNHE1.By surface plasmon resonance（SPR）test,the binding affinity of JL3-1-gp85 and HPRS-103-gp85 ^{E114-/R117G/N123I} to ch NHE1 was about 2-fold of HPRS-103-gp85’s.These results suggested that the binding affinity of JL3-1-gp85 to chNHE1 was higher than HPRS-103-gp85’s and the mutation of amino acid 114,117 and 123 on gp85 enhanced the binding capacity to ch NHE1.In this study,for the first time,we identified the receptor-binding domain of ALV-J gp85.Moreover,we found that the binding capacity of JL08CH3-1 gp85 with chNHE1 was higher than HPRS-103 gp85 and the 114,117 and 123 amino acid residues on gp85 was crucial to receptor binding capacity.These findings lay the foundation for the research of ALV-J infection and pathogenic mechanism,and also provide a new idea for virus replication ability and pathogenicity of ALV-J.