BmFoxO Gene Promotes Anti-BmNPV Immune Responses In Bombyx Mori

As the main spinning insect in China’s silk industry,silkworm will be attacked by various pathogens during its growth and development,such as bacteria and viruses.Among the various viruses,the Bombyx mori nucleopolyhedrovirus（BmNPV）has caused the most damage to the silkworm.In recent years,a variety of immune pathways have been reported to participate in the immune process of insects,such as JAK/STAT pathway,RNAi pathway,Imd pathway,and insulin signaling pathway（ILS）.When the metabolic reaction occurs in the insect body,the ILS pathway is activated to regulate the metabolism of glycolipids.In addition,the ILS pathway also plays a very important role in immunity.Studies have found that when insects affected by starvation,they would activated the FoxO protein in the ILS pathway to regulate the expression of antimicrobial peptide genes to resist microbial infections,and when they were infected by AcMNPV viruses,PI3K-Akt pathway in the ILS pathway was activated to promote virus multiplication.FoxO belongs to the O subfamily member of the Forkhead transcription factor family.It exists in a variety of eukaryotic organisms and participates in a variety of biological pathways,including cell cycle,antioxidant stress,genomic repair,metabolism,immunity and apoptosis and autologous.FoxO is a transcription factor located downstream of the PI3K-Akt pathway in the ILS pathway,and its activity is inhibited by the PI3K-Akt pathway.Studies have found that Drosophila FoxO（dFoxO）could enhance the RNAi immune pathway and significantly inhibited RNA viruses;mammalian FoxO4 was also inhibited the promoter activity of hepatitis B virus（HBV）to inhibit the multiplication of HBV,these studies showed that FoxO can participate in the immune process after virus infection.There is only one kind of FoxO in the silkworm.However,whether BmFoxO can inhibit the BmNPV virus and its mechanism of action have not been reported.Therefore,this study first clarified the expression of BmFoxO after infected with BmNPV virus,then explored the multiplication of BmNPV virus by changing the expression of BmFoxO at the cellular and individual levels of silkworm,and finally explored the immune mechanism of BmFoxO participated in BmNPV virus multiplication by using transcriptional regulation in BmE cells.The research results obtained are as follows:1.Effect of BmNPV virus on BmFoxO expressionThe silkworm embryo cells（BmE）were infected with BmNPV virus solution carrying green fluorescent protein,we observed the green fluorescence at different time points after infection,and used fluorescence quantitative PCR technology to detect the copy number of BmNPV virus.The results showed that with the time of infection of the virus in the cells increases,the green fluorescence carried by the virus gradually increases,and the cells carrying the green fluorescent protein reach 20%after 24 hours of infection.The intracellular viral reproduction copy number showed a significant difference after 6 hours of infection,after 6h hours it gradually increases with time.After BmE cells and fourth instar silkworm were infected,the cells and silkworm midgut RNA were collected at different time points.The qPCR results showed that BmNPV virus had a significant inhibitory effect on BmFoxO at both the cellular and individual levels.Using RNA double-strand interference to reduce the expression of BmFoxO（dsBmFoxO）,dsBmFoxO was used to treat BmE cells infected with BmNPV virus,and found that BmNPV fluorescence level,virus copy number,transcription level and protein level were enhanced.The above results indicated that BmNPV inhibited the expression of BmFoxO after infection with silkworm,and the reduced BmFoxO expression could enhance the multiplication of BmNPV virus.2.Overexpression of BmFoxO inhibits BmNPV multiplicationIn order to further explore the effect of BmFoxO on BmNPV,we used the cloned BmFoxO to carry out phosphorylation site mutations to obtain BmFoxO^CA.Two forms of BmFoxO overexpression plasmids(OE-BmFoxO,OE-BmFoxO^CA)were transfected into BmE cells and infected with BmNPV after transfected 48 hours later.The multiplication of BmNPV were detected,the results showed that after overexpression of BmFoxO in silkworm embryo cells,it could inhibit the fluorescence level of BmNPV,Virus copy number,transcription level and protein level,indicating that overexpression of BmFoxO significantly inhibited the multiplication of BmNPV.The BmFoxO systemic over-expressed transgenic silkworm was used to infect BmNPV virus at fourth instar.The whole silkworm genome of control group and transgenic silkworm group were extracted 48h after infection to detect the virus copy number,and the mortality of each group after 10days of infection was counted..The results showed that after infection with the virus,the viral copy number of the transgenic silkwormgroup was 40%lower than that the control group,and the statistical mortality results showed that mortality of infected silkworms in transgenic silkworm group was reduced by 14%with the control group.The above results indicated that BmFoxO overexpression had a significant inhibitory effection on BmNPV virus,and could reduce the mortality of diseased silkworms in silkworm after BmNPV infection.3.The immune mechanism of BmFoxO inhibits BmNPV multiplicationIt has been found that the infection of AcMNPV could enhance the level of p-Akt protein,and p-Akt had a strong phosphorylation inhibition effect on BmFoxO.We collected total cell protein to detect the effect of BmNPV on p-Akt protein level at different time points after infection.The results showed that BmNPV virus infectionincreased p-Akt protein level.Therefore,we speculated that BmNPV inhibited the expression of BmFoxO by enhancing p-AKT protein level.In order to explore the related mechanism of BmFoxO participating in BmNPV virus immunity,we studied the effect of BmFoxO overexpression on autophagy and apoptosis after BmNPV infection in cells.The results showed that when cell was not infected,overexpression of BmFoxO could weakly enhance the expression of autophagy genes such as ATG6,ATG7,ATG8 and apoptosis genes such as Caspase8,Cytc,and Caspase3,but after infection with BmNPV,it could significantly enhanced the expression of the above related autophagy and apoptosis genes.In order to determine whether the transcription factor BmFoxO participated in BmNPV immunization through BmPEPCK-2,some experiments such as promoter deletion,EMSA,CHIP were used.Furthermore,we changed the expression level of BmPEPCK-2,then tested autophagy-related genes（ATG6/7/8）and apoptosis-related genes（Caspase8/3/1,p53,Cytc）after virus infection.The results showed that BmFoxO induced the up-regulation of BmPEPCK-2 by binding to the ATAAATAAATA sequence at-1162～-1152bp on the BmPEPCK-2 promoter.We also found that overexpression of BmPEPCK-2 promoted the expression of autophagy-related and apoptosis-related genes and knockdown of BmPEPCK-2 inhibited the expression of these genes after virus infection of BmE cells.The above results indicated that BmNPV inhibited the expression of BmFoxO by enhancing p-AKT protein level.Overexpression of BmFoxO induced increased expression of BmPEPCK-2.BmPEPCK-2 promoted the expression of autophagy and apoptosis genes after cells was infected,inhibited BmNPV virus multiplication and enhanced the virus immunity of silkworm.In summary,we found that BmNPV virus inhibited the expression of BmFoxO by enhancing p-Akt protein,and overexpressing BmFoxO could induceBmPEPCK-2up-regulated expression and promote cell autophagy and apoptosis after infected,thereby inhibited the multiplication of BmNPV virus,reduced virus copy number and silkworm mortality.This study not only further enriches our understanding of FoxO’s involvement in viral immune mechanisms,but also provides a basis for using silkworm as a model organism to study insect-virus interactions.