Research Of The Effect Of Env Protein On The Pathogenesis Of Avian Leukosis Virus Subgroup J And Its Molecular Mechanisms

Avian leukosis virus subgroup J(ALV-J)belongs to alpharetrovirus genus,retrovirus family,which is a kind of immunosuppressive virus mainly causing myeloid leukemia and hemangioma in chickens.In 1989,ALV-J was first isolated and identified in the UK,while the first case of ALV-J infection in China was reported in 1999.ALV-J possesses higher pathogenicity,transmission ability and is more dangerous comparing with the other ALV subgroups.At present,ALV-J infection has been prevalent worldwide,which has brought huge economic losses to the poultry industry.However,there are no effective antiviral drugs and vaccines for ALV-J infection.Detection and eradication of core chicken flocks is the main strategy to prevent and control ALV-J infection.Therefore,studying on the pathogenesis of ALV-J and exploring the host factors of anti ALV-J infection will be benefit for developing anti-virus drugs for ALV-J and new strategies for breeding chickens resistant to ALV-J.Env protein is thought to be closely related to ALV-J infection and its pathogenicity,but little is known about its specific role and molecular basis in ALV-J pathogenesis and oncogenesis.In this study,we studied the effect of ALV-J Env protein on the pathogenicity of ALV-J and its molecular mechanism.1.Construction and rescuing of recombinant viruses expressing different C-termini of Gp37 proteinOur previous studies found that ALV-J Env can be divided into three types:inhibitory Env,active Env and bi-functional Env according to the cytoplasmic domain(CTD)within Gp37.To explore the effect of three types of Env and the tyrosine motifs in the CTD on the pathogenicity of ALV-J,ALV-J infectious clones with different C-terminus and tyrosine site mutants in Gp37 were constructed by homologous recombination technology using J1 infectious clone(bi-functional Env)as backbone.Total of nine novel infectious clones were constructed and designated as EAV-HP(inhibitory Env),NY-EAV-HP,YF-EAV-HP,4817(active Env),YN-4817,2YF-4817 and NFF-4817,YF-J1,YN-J1,respectively.J1 and the constructed infectious clones were then transfected into DF-1 cells following with five serial passages and we successfully rescued all the recombinant ALV-J,which laid the foundation for further exploring the effect of different types of Env and tyrosine motif in Gp37 on the pathogenicity of ALV-J.2.Gp37 regulates the pathogenesis of ALV-J via its C-terminusTo evaluate the replication kinetic of the ten ALV-J rescued viruses expressing different C-terminus of Gp37 in vitro,all the recombinant viruses were respectively inoculated into DF-1 cells to generate their viral growth curves.Viral growth kinetics did not only indicate ALV-J with active env is the fastest in replication and ALV-J with inhibitory env is the lowest among the ALV-J carrying three types of Env,but also indicate that the tyrosine sites essentially affected the replication of ALV-J in vitro.To evaluate the pathogenicity of ALV-J with three different types of Env,1-day-old SPF chickens were inoculated with EAV-HP,4817 and J1 rescued viruses,respectively,and the viremia,cloacal viral shedding,organ tissue viral load,antibody production and body weight change of the chickens were monitored.The results showed that the chickens infected by ALV-J with active or bi-functional env showed higher viremia,cloacal viral shedding and viral tissue load compared with those infected by ALV-J with inhibitory env.In addition,the body weight of EAV-HP group was lower than the PBS group,however the body weight of 4817 or J1 group decreased more severely compared with EAV-HP group.Interestingly,the overall antibody positive rate and titer for EAV-HP group were both higher than those of 4817 and J1 groups in the detection of antibodies against ALV and ALV-P27 antigen.Therefore,the C-terminus of Gp37 regulates the pathogenesis of ALV-J,and the pathogenicity of ALV-J containing active or bi-functional Env is higher than that of ALV-J containing inhibitory Env.3.ALV-J and its Env induce dephosphorylation of SHP-2The CTD of ALV-J Gp37 contains different tyrosine motifs,in which both inhibitory Env and bi-functional Env contain ITIM that can recruit SHP-2,which suggests that ALV-J may participate in SHP-2-mediated signaling pathway.To investigate the effect of ALV-J on SHP-2,DF-1 and HD11 cells were infected with ALV-J GY03,and we found the phosphorylation level at Y542 site in SHP-2 was decreased.In addition,ALV-J Env was further over-expressed in DF-1 cells and HD11 cells.The results showed that Env protein could down-regulate the pathogenesis and oncogenesis of ALV-J,the development of anti ALV drugs and shortening the time line for ALV isolation and identification.