| Infectious bursal disease is an acute and contagious disease caused by infectious bursal disease virus(IBDV),which mainly threatens 3 to 6 weeks chicken by targeting the B-lymphocyte precursors within bursa of Fabricius.IBDV belongs to the family Birnaviridae and its genome consists of segments A and B which encode five viral proteins totally.IBDV infection could alter the expression of interferon stimulated genes(ISGs),but there is still not much work focusing on the interaction between IBDV and ISGs.In the present study,we selected two ISGs(IFIT5 and Viperin)which were up-regulated upon IBDV infection and investigated their effects on IBDV replication.IFIT5 is the sole member of avian IFIT family.Our previous proteomic data showed that IBDV infection significantly induce the expression of chicken IFIT5 in DF-1 cells.However,there is no report on the chicken IFIT5 which is still a putative protein in the Genbank database.In this study,we cloned chicken IFIT5 gene from IBDV-infected DF-1 cells and the full-length of this gene is 1440bp,encoding 480 amino acids.Bioinformatics analysis showed that the protein possessed six canonical TPR motifs and shared the highest amino acid identity with turkey but the lowest identity with fish.Using the polyclonal antibody against IFIT5 which was generated by using the prokaryotic IFIT5 protein as antigen,we demonstrated that both the mRNA and protein levels of chicken IFIT5 were up-regulated upon poly(I:C),IFN-a and IFN-? stimulation and certified the interaction between chicken IFIT5 and Mda5.Furthermore,the transcription level of IFN-? was decreased when chicken IFIT5 was knockdown.To illuminate the relationship between chicken IFIT5 and IBDV infection,the dynamic expression of chicken IFIT5 was analyzed after IBDV infection.The results revealed that chicken IFIT5 could be induced by IBDV infection in vitro and vivo in a dose-dependent manner.Moreover,we proved that IBDV-encoded VP1 and genome dsRNA were the viral components stimulating the chicken IFIT5 expression.However,overexpression and knockdown of chicken IFIT5 showed no effect on IBDV replication in DF-1 cells,suggested that IBDV might escape the antiviral function of chicken IFIT5 by the unknown mechanism.As another ISG with antiviral activity,Viperin was also up-regulated upon IBDV infection.We also examined the effect of Viperin on IBDV replication.In accordance with IFIT5,overexpression of chicken Viperin failed to change the viral protein expression and viral titer of IBDV in DF-1 cells,but surprisingly,human Viperin could inhibite the replication of IBDV in HEK293T cells obviously.Furthermore,we found that Viperin was capable of eliciting cellular apoptosis but not autophagy,inferred that human Viperin restricted IBDV infection by enhancing cellular apoptosis.Besides innate immunity,autophagy is also a pathway to limit viral infection and viruses had evolved strategies to resist it.In the present study,we investigated the regulation of VP4 on autophagy.Although no interaction between VP4 and LC3 was detected,we still observed autophagosomes surrounded by VP4 and exogenous expression of VP4 could increase the conversion of LC3-I to LC3-II and accumulation of P62,indicating that autophagy was inhibited by VP4.We also proved the co-localization and interaction between VP4 and Beclin-1,which is a crucial factor in autophagy pathway,and assumed that VP4 could block the fusion of autophagosomes and lysosomes or degradation of autophagosomes in lysosomes by targeting Beclin-1.Furthermore,our finding also showed that VP4 with deletion of two C-teminal residues could induce apoptosis which related to its protease activity. |
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