Autophagy Promotes The Release Of Infectious Bursal Disease Virus

Infectious bursal disease is an immunosuppressive disease of young chickens that causes considerable economic loss to the poultry industry worldwide.The causative agent,infectious bursal disease virus(IBDV),belongs to the genus Avibirnavirus of the family Birnaviridae.IBDV is the most extensively studied virus in this family in terms of our collective understanding of its molecular and cellular biology,structure,and pathogenesis;therefore,because the autophagic pathway and its function during dsRNA virus infection are poorly understood,IBDV is an important model system for dsRNA virus.The Avibirnavirus VP2 binds to the pathogen receptor HSP90AA1 and induces autophagy by inactivating the AKT-mTOR pathway during the early stage of virus infection.The relationship between autophagic signaling and viral infection,the autophagic flux induced by IBDV during the late stage of virus infection,and the mechanism by which autophagy affects viral replication require further investigation.The Birnaviridae family viruses integrate their genome into a complex structure known as the “viral factory”,which localises to the cytoplasmic face of membrane of endocytic compartments.Additionally,the Golgi apparatus plays an important role in IBDV assembly.However,the mechanism of viral maturation and release needs further investigation.A recent study indicated that exocytosis of varicella-zoster virus virions involves a convergence of endosomal and autophagy pathways.Thus,in this study,the cellular aspects participating to the birnaviral replication process were studied to gain insight into whether autophagy pathway is involved in viral maturation and release.In this study,infectious bursal disease virus(IBDV)was used to investigate the role of autophagy in Avibirnavirus replication.We demonstrated IBDV induction of autophagy as a significant increase in puncta of LC3+ autophagosomes,endogenous levels of LC3-II,and ultrastructural characteristics typical of autophagosomes during the late stage of infection.Cells were starved of amino acids,a condition known to induce autophagy.Viral proteins pVP2 and VP2 levels significantly increased with starvation,and the infectious progeny virus titres,both intracellular and extracellular,significantly increased with autophagy induction by nutrient deprivation.DF-1 cells infected with IBDV were treated with wortmannin(WM),a PI3 kinase/Ptd Ins 3-kinase inhibitor known to inhibit autophagosome formation,which significantly reduced the intracellular and extracellular viral titres.To verify that autophagy inhibition impairs IBDV replication,cells infected with IBDV were treated with 3-MA in nutrient deprived condition,which reduces autophagy signaling by inhibiting type-III PI-3 kinase activity.Treatment with 3-MA reduced viral protein pVP2 and VP2 levels,moreover,significantly reduced both intracellular and extracellular viral titres.Viral protein p VP2 levels exhibited a significant decrease in LC3 knocked-down cells and the infectious progeny viruses both in intracellular and extracellular were significantly reduced by LC3 B shRNA versus scrambled shRNA.ATG5 was effectively knocked down and the level of LC3-II was also reduced obivously,which indicated that the autophagic signaling was effective knocked down based on ATG5 silencing.The results showed that the level of pVP2 significantly decreased compared to that with the scrambled siRNA.The infectious progeny virus titres,both intracellular and extracellular,were reduced significantly in ATG5 knockdown cells.In mock cells,LAMP1 was distributed homogeneously throughout the cells at a low level.However,in virus-infected cells,LAMP1 was much more concentrated and presented as spots around the viral components(p)VP2,VP3,and dsRNA,with partial co-localisation.The results showed that both the glycosylated LAMP1 and the polypeptide core increased in IBDV-infected cells relative to the proteins in mock cells.BAF A1 treatment resulted in inhibition autophagosomes fusion with lysosomes,with increased levels of LC3-II and p62.Viral proteins pVP2 and VP2 showed a significant decrease in the presence of BAF A1.Inhibition of autophagosome fusion significantly reduced the infectious progeny virus titres both intracellularly and extracellularly.Cargo is hydrolysed in the acidic lysosomes.Chloroquine(CQ)inhibits lysosome acidification,thereby inhibiting the hydrolysis activity of lysosomes.The amount of pVP2 significantly increased with CQ treatment,whereas the amount of VP2 significantly decreased in CQ-treated cells,suggesting that maturation of VP2 derived from pVP2 decreased because of the loss of lysosomal hydrolysis activity.The infectious progeny virus titres both in the supernatant and in cell cultures were significantly reduced in CQ-treated versus untreated cells.Thus,IBDV infection induces amphisomes and/or autolysosomes formation,but does not lead to active degradation.Viral maturation is enhanced by the low pH environment of amphisomes/autolysosomes.Using immuno-transmission electron microscopy(TEM),we observed that a large number of intact IBDV virions were arranged in a lattice surrounded by p62 proteins,some of which laid between virions.Additionally,many virions were encapsulated within the vesicular membranes with an obvious release stage observed by TEM.The autophagic endosomal pathway facilitates low pH-mediated maturation of viral proteins and membrane-mediated release of progeny virions.The present study demonstrated that autophagy enhances viral replication at the late stage of infection,and the autophagy pathway facilitates IBDV replication complex function and virus assembly,which is critical to completion of the virus life cycle.Moreover,the virus hijacks the autophagic vacuoles to mature in an acidic environment and release progeny virions in a membrane-mediated cell-to-cell manner.This autophagic endosomal pathway is proposed as a new mechanism that facilitates IBDV maturation,release,and re-internalisation.