Preliminary Study On The Interaction Between Avian Reovirus ?A Protein And Host Proteins HSP70,HSP40 And RPL4

Avian reovirus(ARV)is a member of the Orthoreovirus branch of Reoviridaeae.ARV are primarily causes viral arthritis/tenosynovitis,dwarfism,etc in poultry.In addition,it also causes immunosuppression,decreased performance and death.ARV infection is therefore extremely harmful to the poultry industries and can cause huge economic losses.ARV is a double-stranded RNA virus that does not contain a capsule.Its genome consists of 10 RNA gene fragments,encoding eight viral structural proteins(?A,?B,?C,?A,?B,?A,?B,and ?C)and four non-structural proteins(?NS,?NS,p10 and p17).The ?A protein is a structural protein encoded by the S2 gene between the outer capsid ?BC protein and the underwear shell ?A protein,which promotes the stability of the inner and outer capsid structures of ARV.The ?A protein can inhibit the activation process of the protease by binding to the dsRNA sequence,which can inhibit the secretion of interferon in the cell,thereby assisting the virion to evade the immune defense in the host cell.In the process of virus infection,protein A can provide energy for viral transcription and replication through the activity of its hydrolase;The ?A protein also regulates the signaling pathways in the host cell and promotes replication of the virus in the host cell;In addition,the protein plays an important role in the assembly of virions.In the virus-infected cells,there is a certain relationship between the virus-encoded protein and the host protein,which affects the infection process of the virus.Therefore,the study of ARV a A protein and its interaction protein is extremely important,and can further understand the pathogenesis of the virus.HSP70,HSP40,and RPL4 proteins are host proteins that are highly conserved and have important biological roles in host cells.HSP70 and HSP40 proteins have the function of chaperones,and there are many studies on the interaction between these two proteins and viral proteins.It is known that pig-derived HSP70 and human HSP40 proteins can ensure the correct folding and assembly of proteins during virus invasion and promote the replication and proliferation of viruses in vivo.RPL4 protein can participate in the assembly process of ribosomes,and avian RPL4 protein can participate in the process of virus invasion of the host cells.In our laboratory,proteomics and yeast two-hybrid studies have found that HSP70,HSP40 and RPL4 proteins are associated with ARV a A protein,therefore,these three proteins were selected to study the host proteins that interact with ARV ?A protein.The eukaryotic expression recombinant plasmids pEF1?-HA-?A,pEF1?-Myc-HSP70,pEF1?-Myc-HSP40(DNAJB6)and pEF1?-Myc-RPL4 were constructed and verified by colony PCR,double enzyme digestion and sequencing.The results showed that the recombinant plasmids of pEF1?-HA-?A,pEF1?-Myc-HSP70,pEF1?-Myc-HSP40(DNAJB6)and pEF1?-Myc-RPL4 were successfully constructed.The constructed eukaryotic expression recombinant plasmid was transfected into human embryonic kidney cell(HEK293T)cells to express the protein,and verified by indirect immunofluorescence and Western blot.The results showed that HA-?A,Myc-HSP70,Myc-HSP40(DNAJB6)and Myc-RPL4 fusion proteins could be correctly expressed in HEK293T cells.The pEF1?-HA-?A eukaryotic expression recombinant plasmid was co-expressed with pEF1?-Myc-HSP70,pEF1?-Myc-HSP40(DNAJB6)and pEF1?-Myc-RPL4 eukaiyotic expression recombinant plasmids in HEK293T cells,respectively.The interaction was verified by immunoprecipitation experiments.The results of co-immunoprecipitation showed that the interaction between ARV ?A protein and avian source HSP70 and RPL4 protein was negative.The ARV ?A protein and avian source HSP40(DNAJB6)protein have a one-way interaction positive band.That is,when co-immunoprecipitation was carried out using a rabbit-derived HA-tagged antibody as a primary antibody,a positive band was detected by Western blot detection using a murine Myc-tagged antibody as a primary antibody,and no significant band was detected by a reverse immunoprecipitation test.This study speculates that there is an interaction between ARV ?A protein and avian HSP40(DNAJB6)protein,which needs further research.This study have created conditions for the further exploration of the pathogenesis of ARV.