Receptor Function Domain Identification Of Subgroup J Avian Leukosis Virus And Comparison Among Different Chicken Species

Subgroup J avian leukosis, caused by subgroup J avian leukosis virus(ALV-J), is a neoplastic disease characterized by excessive proliferation of cell components in poultry hematopoietic tissue. In recent years, ALV-J spread widely among local chicken species in China, causing serious financial losses. The process of retrovirus infection began with the recognition of viral envelop protein to its cellular receptor, followed by interaction between and finally the entrance of virus into the host cells. Different subgroup viruses use different cellular receptors. Previous study proved that, chicken Na+/H+ exchanger type I(ch NHE1) is cellular receptor for ALV-J. NHE1 is a house-keeping gene that exists on the eukaryotic animal’s cell membrane. Under low pH, the envelop of ALV-J recognizes chNHE1, followed by conformational change of the transmembrane part, which prompts the fusion of envelop protein and cell membrane, and finally the entry of virus into cells. The binding of virus and its receptor is the first step of viral entry and infection, which possess crucial significance. The study of interaction between virus and its receptor benefits to the exploration of virus infection and pathogenic mechanism.We firstly performed the expression and purification of soluble gp85 protein by co-expression with a secreting signal peptides, so as to establish the flow cytometry analysis method of testing receptor binding ability. Besides, we also constructed and rescued a RCAS virus with a luciferase maker protein and ALV-J envelop. Based on this, a method for detecting viral entry level was established. Then, we exchanged the 6 extracellular loops(ECLs) to the corresponding ECLs on human NHE1 and to the nonfunctional HA tag, only to find that, ECL1 and ECL3 of ch NHE1 influenced receptor function. By segment replacement, we identified amino acids 28 to 39 to be the minimum segment that influenced receptor function. Next, we performed site mutations of 12 amino acids, and finally identified 30, 33, 34, 38, 39 amino acids to be the key sites that influence receptor function. Similarly, by segment replacement of ECL3, we identified 214/215 amino acids showed synergistic effect on influence receptor function. In the following gain of function assay, we performed amino acid and segment exchange of human NHE1 and chNHE1 on the backbone of human NHE1. Results showed that, amino acid 28 to 39 is the minimum segment which gives receptor function to human NHE1. This further verified that 28-39 amino acids is the key domains during the binding between gp85 and chNHE1.Epidemiological survey showed that, the ALV-J infection rates of different local chicken species are significantly different. To verify whether this difference is associated with domain mutation of chNHE1, this study selected 18 local chicken species from four main chicken breeding province and analyzed the sequences of chNHE1 domain,only to find that the receptor genes are completely consistent and highly conservative.This study for the first time identified the key amino acids and segments of chNHE1 in receptor function and at the same time discovered the high conservation of chNHE1 function domains. These findings lay the foundation for the research of ALV-J infection and pathogenic mechanism, and also provides a new idea for the prevention and control of ALV-J.