Interaction Mechanism Between Subgroup J Avian Leucosis Virus And Host Cells

Avian leucosis virus subgroup J(ALV-J)belongs to C type avian retrovirus,retroviridae.The infection of ALV-J would result avian leucosis disease,an oncogenic and immunosuppressive avian diease that is similar to Marek virus(MDV)disease and Reticuloendotheliosis virus(REV)disease.And the symptoms include myeloma,hemangiomas and leiomyomas,thus poses great threat to avian industry.Research on ALV-J's pathogenic mechanism is a critical part of the prevention against ALV-J,especially the research on ALV-J's invasion into susceptible host cells and how it evades innate immunity to establish infection.Our work focuses on these two parts and tries to shed a light on mechanism of avian virus' immunity suppression,discover potential medicine therapeutic targets,expanse molecular tools for avian retrovirus research and lay down foundation for future research.1.GRP78's role in ALV-J's invasion into host cellsOur lab identified chANXA2 as a novel host receptor for ALV-J's infection.Meanwhile,another protein,chGRP78,was also identified to be able to interact with ALV-J's envelope protein.As a well known heat shock protein,GRP78 has been reported to critical for some other virus' replication.Our previous data indicates that the block of membrane abound chGRP78 with antibody could effectively inhibit ALV-J's infection on DF1 cells.In this research,we employed siRNA which specifically against chGRP78's mRNA.The replication of ALV-J on DFl cells was significantly inhibited after chGRP78's expression level was reduced around 65%by siRNA.On the other hand,when ALV-J unsusceptible cell 293T was transfect with increasing amount of eukaryotic expression plasmid which harbors chGRP78's CDS,the invasion of ALV-J was also increased,which has a positive correlation with transfection.Experiment on GEF,an ALV-J unsusceptible avian cell,further confirms that transfection of chGRP78 would convert it into susceptible cells.In conclusion,chGRP78 plays an important role as invasion intruducer in ALV-J's infection on host cells.2.Toll like receptors'(TLRs)role during ALV-J infectionBesides chGRP78,role of receptors related to host innate immunity in ALV-J infection was also explored.We used the ligands against TLR2-5,7 and 21 to stimulate CEF for 16 h respectively,until the infection is over.Then at 12,24,48 and 72 h post infection(h.p.i),the CEF was stimulated again with those ligands.Meanwhile,real-time PCR,IFA,WB and TCID50 assay were employed to test ALV-J's replication.The results indicate that ALV-J's infection was inhibited at any time point when TLR2-4 was stimulated.Stimulation of TLR7 and 21 before ALV-J infection or at 12hpi and 24hpi would inhibit ALV-J's replication.While only stimulation of TLR5 at 12h post infection time points would inhibit ALV-J's replication.In conclusion,regardless of time points,stimulation of all the TLRs above would inhibit ALV-J's replication on CEF cell,only the time when the ligands stimulated is different.This indicates that the activated innate immunity response could effectively inhibit ALV-J's infection.Vise versa,ALV-J probably establish the infection through interaction of the innate immunity pathway.3.MyD88 independent pathway is activated during ALV-J's infection in CEF cellsIn this part,we further explore with which pathway ALV-J interact to establish the infection.Based on the specificity of TLR's ligands repertoire,we assume that ALV-J probably interacts with TLR2-4,TLR7 and TLR21.We infected CEF with purified ALV-J virion and detected the activation of each pathway with real-time PCR at different time points post infection.The results indicate that at 72 and 96 h post infection,only TLR3 was activated.Since TLR3-activated pathway is independent of MyD88,we then detected the activation of MDA5,the downstream molecular of RIG-1 pathway(MyD88 independent).It showed that ALV-J infection would also upregulate mRNA of MDA5,which further confirms ALV-J's interaction with MyD88 independent pathway.Moreover,real-time PCR revealed that ALV-J's infection also upregulate mRNA of IRF3/7,the transcript factors which lie in the very downstream of TLR3 pathway.While the IFN-? and IFN-? were not upregulated.This indicates host cell's innate immunity could effectively response to ALV-J;however,ALV-J's anti innate immunity capacity blocked IRF3/7's function.4.Chicken IRF3/7's role in innate immunity against ALV-J infectionIn this research,we expressed chIRF3/7 with prokaryotic expression vector pCold-TF.Then we used purified recombinant protein as antigen to produce poly anti-serum in 5-week old Balbc/c mouse.The acquired anti-serum could recognize chIRF3/7 specifically and effectively.We also transfect CEF with pcDNA3.1-chIRF3/7 to over express chIRF3/7.Real-time PCR,WB and TCID50 assay all indicate that ALV-J's replication was significantly inhibited after transfection.Meanwhile,overexpression of chIRF3/7 significantly restore IFN-?'s mRNA level.This strongly indicates that ALV-J inhibit host's innate immunity through block of chIRF3/7.5.Preliminary research on ALV-J Env protein's role in anti innate immunityThe low homology of Env gene makes ALV-J different from other subgroups of ALV.Some scholars come up with the theory that Env's coding protein is responsible for ALV-J's tumorigenicity and pathogenicity.Based on this,we assume that Env gene is probably also responsible for ALV-J's anti innate immunity capacity.When DF1 cells are transfect with pcDNA3.1-Env,both real-time PCR and dual-luciferin assay shows that Env gene could inhibit IFN-? 's mRNA level at 1,6 and 12 h post stimulation with poly(I:C).At 12 h post stimulation,although the mRNA of TLR3 is upregulated,both IRF3/7 and IFN-? 's mRNA are down regulated.Based on this,we assume that Env gene may anti innate immunity during ALV-J infection,and it is closely related to the block of IRF3/7's function.