Molecular Mechanism Of Bombyx Mori Cypovirus-induced Autophagy

Bombyx mori cypovirus(BmCPV)belongs to Reovirus.BmCPV can infect midgut cells of B.mori causing cytoplasmic polyhedrosis,causing cocoon crop failure.Eukaryotes keep cell homeostasis and resist pathogens infection through autophagy pathway.So far,the mechanism of interaction between BmCPV infection and host cell defense is unclear.This study about the mechanism of interaction between BmCPV infection and autophagy has a great significance for understanding the the mechanism of interaction between BmCPV infection and host cell defense.1.BmCPV infection can induce autophagy in silkworm cellsIn this study,the pathological changes of midgut cells after BmCPV infection were observed by TEM(transmission electron microscope),and autophagosome-like structures appeared in infected cells.LC3 is the abbreviation of microtubule-associated proteins light chain 3(MAP1LC3).LC3 can mark the formation of autophagosomes and is used as a marker for detecting autophagy.When pIZ-EGFP-LC3 plasmid was transfected into BmN(B.mori ovarian)cells,this plasmid could mark the formation of autophagosomes.BmCPV infection can significantly increase the number of autophagosomes.Western Blotting results showed that the level of LC3-II protein was importantly increased in BmN cells and silkworm midgut after BmCPV infection.These results indicate that BmCPV infection can induce autophagy in silkworm cells.2,BmCPV infection induces mitochondrial autophagyIn order to investigate the type of autophagy induced by BmCPV infection,TEM was applied to observe the BmCPV infected silkworm midgut.Damaged mitochondria and mitochondrial-autophagosome structures appeared in BmCPV infected midgut cells.Specific antibodies were used to detect the proteins related to PINK1-Parkin mitochondrial autophagy(mitophagy)signaling pathway in BmCPV-infected BmN cells.Western Blotting results showed that the levels of PINK1,Parkin,and P-Drpl proteins were importantly increased,and the level of Tom20 protein was oppositely decreased in BmCPV-infected BmN cells.Furthermore,after the expression of Parkin gene was silenced by siRNA,the level of Parkin/α-tubulin decreased importantly,and the level of LC3-II/a-tubulin decreased accordingly.After the expression of Drpl gene was silenced by siRNA,the level of P-Drpl/α-tubulin decreased importantly,and the level of LC3-II/α-tubulin decreased accordingly.These results indicate that BmCPV infection induces mitophagy,and blocked mitophagy pathway will inhibit autophagy induced by BmCPV infection.3.Autophagy promotes BmCPV proliferationTo research the interaction between BmCPV infection and mitophagy,BmN cells treated with the autophagy inducer rapamycin(Rap)and the autophagy inhibitor 3-methyladenine(3-MA)were infected with BmCPV.When BmN cells were treated with Rap,Western Blotting results showed that the level of BmCPV structural protein VP4/α-tubulin was importantly increased and qPCR results showed the level of BmCPV vp1 gene was accordingly increased.While BmN cells were treated with 3-MA,Western Blotting and qPCR results were opposite.These results indicate that the autophagy induced by BmCPV infection could promote viral proliferation.4.BmCPV encoding structural protein VP4 is a key protein that induces autophagyIn order to investigate key viral proteins that induce autophagy,LC3-EGFP-mCherry plasmid was applied to mark autophagosomes.pIZ-LC3-EGFP-mCherry BmN cells were transfected with different viral protein expression plasmids of pIZT-CSI-pIZT-CS10.Results showed that viral VP4 protein can significantly increase the number of autophagosomes.Furthermore,Western Blotting results showed that the levels of PINK1,Parkin,and P-Drpl proteins were importantly increased,and the level of Tom20 protein was oppositely decreased in VP4 overexpressed BmN cells.These results consistent with the results in BmCPV-infected BmN cells.To summarize,BmCPV encoding structural protein VP4 is one of the key proteins that induces mitophagy.