Study On Autophagy Degradation Of Avibirnavirus VP2 Protein Mediated By Autophagy Receptor P62

Infectious bursal disease virus(IBDV)is a non-enveloped virus,which is an Avibirnavirus belonging to the Birnaviridae family.IBDV genome is composed of two segments of double-stranded RNA A and B.Segment A contains two partially overlapping open reading frames(ORFs),the larger ORF encoding VP2,VP3 and VP4 and the smaller ORF encoding VP5;segment B encodes VP1.IBDV mainly damages 3-6 weeks young chickens,attacking the developing B-lymphocytes in the bursa of Fabricius(BF)in central immune organ to causes immunosuppression.VP2 protein is the main component of virus capsid protein.And VP2 can activate autophagy and regulate virus replication by binding to the receptor on cell membrane.However,the relationship between VP2 and autophagy has not been studied.It has been reported that the capsid protein of some viruses can be recognized by autophagy receptor for degradation,while it is not clear whether autophagy can recognize and degrade the capsid protein VP2 of IBDV.In this paper,we study and analyze the molecular mechanism of p62-mediated selective autophagic degradation of VP2,and further analyze the role of p62 in the process of virus replication.1.Study on the degradation pathway of IBDV VP2 proteinVP2 can be degraded in lysosomes.We detect the expression level of VP2 using autophagy activators or inhibitors to activate or inhibit the autophagy level in cells to further verify the degradation mechanism of VP2.We found that autophagy activators rapamycin and early’s balanced salt solution(EBSS)significantly promoted the degradation of VP2 protein,while autophagy inhibitors wortmannin and chloroquine significantly inhibited the degradation of VP2 protein.These results indicate that VP2 protein can be degraded by the autophagy pathway.2.Study on the degradation of capsid proteinVP2 mediated by autophagy receptor p62At present,selective autophagy is widely studied about autophagy degradation.Among them,autophagy receptor p62 is one of the most studied selective autophagy receptors.It can recognize the substrates through the ubiquitin chain or directly recognize the substrate not modified by ubiquitin.We have confirmed that VP2 can be degraded via the autophagy pathway,and it was reported that p62 mediates the autophagy degradation of viral capsid protein.Therefore,we investigated the relationship between IBDV capsid protein VP2 and autophagy receptor p62.Through immunocoprecipitation and indirect immunofluorescence,we showed that there was an interaction between VP2 and p62.In addition,p62 mediated the co-localization of VP2 and LC3.Meanwhile,the activation of autophagy promoted the interaction between VP2 and p62.Above all was suggested that p62 as an autophagic receptor mediates the selective autophagic degradation of VP2.In order to further explore the mechanism of p62-mediated degradation of VP2,we validated the effect of p62 protein on the degradation level of VP2 protein in p62 knockout cells.We tested the affection of p62 on the degradation of capsid protein VP2 protein in p62-knockout 293T cells.We found that the degradation of VP2 was significantly inhibited in p62 knockout 293T cells compared to wild-type cells,while the degradation of VP2 was importanly promoted when p62 was overexpressed.These results confirmed that p62 mediates autophagic degradation of VP2.Furthermore,we found that the UBA domain is the key domain in the interaction between VP2 and p62.And the UBA domain and the LIR domain(LC3 interacting region,LIR),which are the interaction domains of p62 with substrates and LC3,respectively,are required for autophagic degradation of VP2.3.Study on the degradation of capsid proteinVP2 mediated by ubiquitinationIn selective autophagy,the UBA domain is necessary for p62 to recognize the ubiquitin chains of substrate proteins.We had demonstrated that the UBA domain of p62 mediates the recognition and degradation of VP2.Through the ubiquitination assay,we found that substrate protein VP2 underwent the K63 ubiquitination predominantly.And we further found that the the mutation of 411 lysine(K)to arginine(R)in VP2 abrogated the ubiquitination and the interaction between VP2 and p62 could not occur,which indicated that the ubiquitination of K411 determined the interaction between VP2 and p62.The above results showed that the ubiquitination of K at 411 of VP2 protein and the UBA domain of p62 protein determined the interaction between VP2 and p62,which proved that p62 recognized VP2 through ubiquitin chains.The degradation of VP2 was no longer affected by p62 when K at 411 was mutated into R.These results suggested that the selective autophagic degradation of VP2 protein mediated by p62 is dependent on the ubiquitination of K411 of VP2 protein.4.The effect of p62 on the replication of IBDVVP2 is the main structural protein and the capsid protein of IBDV,whether p62-mediated autophagic degradation of VP2 would affect the replication of IBDV was further studied.We detected the changes of viral protein and viral titer of IBDV in the case of the deletion or overexpression of p62.The results showed that the knockout of p62 promoted the replication of IBDV,and the overexpression of p62 inhibited the replication of IBDV.These results suggested that when the host cell is attacked by IBDV,cells can remove viral component to inhibit the replication of IBDV through autophagy degradation,so as to protect the host from invasion of virus...