Molecular Mechanisms Of Autophagy Induced By ORFV In OFTu Cells

Orf virus(ORFV),an important zoonotic pathogen of animal origin,which not only causes serious harm to sheep production,but also poses a huge hidden danger to human public health security.ORFV has a complex immune escape mechanism,and the inhibition/antagonism of the host antiviral immune response may be a significant cause for its persistent/repeated infection.Although some progress has been made in the mechanism of ORFV immune escape,the molecular mechanism remains to be explored due to the huge ORFV genome.As an important host self-protection and defense mechanism,autophagy plays acrucial role in anti-infection immunity,can not only directly clear pathogens,but also participate in the regulation of innate and adaptive immunity.However,pathogens also evolve a series of strategies to escape or utilize autophagy in the battle between the host and pathogen,which is conducive to the survival of pathogens.Hence,starts from the interaction between ORFV and autophagy,this study focused on the autophagy response and related mechanisms after ORFV infection of OFTu cells,and the impact of ORFV-induced autophagy on virus replication.Firstly,OFTu cells were infected with ORFV at MOI=10,Western blotting,fluorescence microscopy and transmission electron microscopy(TEM)were used to detect the type conversion and localization of microtubule associated protein 1 light chain 3?(MAP1LC3B),and to observe the ultrastructure of autophagosomes.The results demonstated LC3 type conversion and autophagosome formation in OFTu cells induced by ORFV infection.However,autophagy is a dynamic process by which the accumulation of LC3II may be caused by either the increased formation of autophagosomes or the blocked fusion and degradation of autophagosomes and lysosomes.Therefore,to analyze the variation of autophagy flux,we further treated OFTu cells with the autophagy activator Rapamycin(Rapa),the autophagy inhibitor 3-methyladenine(3-MA)and the late autophagy inhibitor Bafilomycin A1(Baf A1),which can inhibit the fusion of autophagosome and lysosome,respectively;The expression of LC3 and the autophagy substrate SQSTM1/P62 under different autophagy states were detected by Western blotting,meanwhile,the activation of autophagy flux was observed by fluorescence microscope.The results indicate that ORFV infection could induce complete autophagy and autophagy flux was activated in OFTu cells.The canonical autophagy pathway is regulated by mammalian target of rapamycin complex 1(mTORC1)and its upstream adenosine-5'-monophosphate(AMP)-activated protein kinase,serine/threonine kinase(AKT)and mitogen-activated protein kinase(MAPK)signaling cascades through its phosphorylation of Unc-51-like kinase 1(ULK1)and other non-autophagy-related gene.To investigate the relationship between ORFV-induced autophagy and mTOR cascade signals in OFTu cells,the expression of mTOR,the key protein of autophagy pathway,and its negative regulator,nodular sclerosis complex 2(TSC2)were detected by Western blotting.The results showed that TSC2 was activated by phosphorylation,while mTOR activity was inhibited as ORFV infection induced autophagy in OFTu cells,suggested that mTOR signaling pathway was involved in this autophagy process.To further clarify the pathway and mechanism of ORFV-induced autophagy,OFTu cells were treated with AMPK specific activator(AICAR)and inhibitor Compound C,Akt specific activator SC79,and ERK1/2 specific inhibitor U0126.After pharmacological treatment,Western blotting was used to measure the autophagy related pathways and key proteins upstreamed mTOR such as AMPK,PI3K/Akt,MAPK/Erk1/2.The results showed that the infection of ORFV could induce autophagy by inhibiting the PI3K/Akt/mTOR signaling pathway or inhibit mTOR activity through activating the MAPK/Erk1/2/mTOR signaling pathway,on the contrary,it has no connection with AMPK/mTOR signaling pathway.In order to research the impact of autophagy activation on the replication and proliferation of ORFV,TCID₅₀ test was used to measure the titer of ORFV under different autophagy states.It showed that the autophagy in OFTu cells induced by ORFV can promote viral replication.Results up to this point will not only enrich the study of virus-host interaction network,but also help to reveal the mechanism of ORFV infection and immune escape,and particularly significant to provide a theoretical basis for a comprehensive analysis of the pathogenesis of ORFV.