Identification And Mechanism Analysisof Anti-BmNPV Antimicrobial Peptides From Silkworm

Bombyx mori,as a model insect of Lepidoptera,has important economic and research value.Like other members of Lepidoptera,the silkworm has innate immune system and lacks acquired immune system.Every year,serious economic losses are caused by silkworm diseases.Bombyx mori nuclear polyhedrosis virus disease caused by Bombyx mori nucleopolyhedrovirus(BmNPV)is particularly harmful to the sericulture industry,which not only reduces the output and quality of silk,but also is not conducive to the sustainable development of the sericulture industry.Therefore,exploring the molecular mechanism of interaction between silkworm and BmNPV and identifying the antiviral proteins of silkworm that can interact with the virus play an important role in the effective prevention and control of the disease.Antimicrobial peptides(AMPs)are a class of small molecular polypeptides with immune effects produced by the coding of specific genes in silkworm.The number of amino acid residues in AMPs is less than 100,which is an important antimicrobial factor in insect humoral immunity.AMPs have the characteristics of small molecular weight,strong thermal stability,broad antimicrobial spectrum,non-immunogenicity and non-toxicity to normal cells of higher animals.Previous studies have shown that silkworm antimicrobial peptides have broad-spectrum antimicrobial activity,but whether they have the ability to resist BmNPV has not been reported.Therefore,this study aimed at identifying the ability of silkworm antimicrobial peptides to resist BmNPV and exploring its resistance mechanism,and obtained the following research results:1.Screening of up-regulated antimicrobial peptides induced by BmNPV virusIn this study,the silkworm BmNPV resistant strain 871 C was used as a model to analyze the antimicrobial peptides up-regulated by BmNPV virus infection.Nine up-regulated antimicrobial peptides genes were obtained by transcriptome data,include BmGloverin1(BmGlv1),BmGloverin3(BmGlv3),BmAttacin1(BmAtt1),BmLebcin3(BmLeb3),BmMoricin3(BmMor3),BmCecropinB1(BmCecB1),BmCecropinB2(BmCecB2),BmCecropinB3(BmCecB3)and BmCecropinD1(BmCecD1).Four up-regulated antimicrobialpeptides were obtained by proteome data,include BmGlv1,BmGlv3,BmGlv4 and BmCecD1.In addition,the expression levels of BmCecB2,BmCecD1,BmGlv3 and BmLeb3 were significantly up-regulated at the silkworm level after infection with BmNPV;the expression levels of BmCecB1,BmCecB2,BmCecD1,BmGlv3 and BmLeb3 were significantly up-regulated after infection with BmNPV at the cell level.Based on these results,BmCecB2,BmCecD1,BmGlv3,and BmLeb3 may be involved in the antiviral pathway.2.Identification of Antimicrobial Peptide Resistance to BmNPV VirusIn order to identify the anti-BmNPV ability of the selected antimicrobial peptides,the four selected antimicrobial peptides had signal peptide and no nuclear localization signal.Secondly,four antimicrobial peptides overexpression vectors pIZ/V5-His-BmGlv3,pIZ/V5-His-BmLeb3,pIZ/V5-His-BmCecB2 and pIZ/V5-His-BmCecD1 were constructed.Overexpression of antimicrobial peptides in cells,the viral replication and proliferation were detected by TCID50,quantitative and flow cytometry.The results showed that the four antimicrobial peptides reduced the viral infection rate compared with the control group,but the degree of reduction was different.The order of virus proliferation was: blank control group > pIZ-BmLeb3 group > pIZ-BmCecB2 group > pIZ-BmGlv3 group > pIZ-BmCecD1 group.We further identified the dose effect between antimicrobial peptides.Compared with the overexpression of BmCecB2 or BmGlv3 alone,the co-overexpression of BmLeb3 and BmCecB2,BmLeb3 and BmGlv3 enhanced their antiviral ability,suggesting that antimicrobial peptides could synergistically enhance their antiviral ability.In order to identify the antiviral ability of antimicrobial peptide in silkworm,baculovirus expression system was used to overexpress antimicrobial peptide gene in silkworm larvea.The results showed that overexpression of four antimicrobial peptides inhibited the proliferation of BmNPV.BmCecD1 had the strongest antimicrobial activity against BmNPV,while BmLeb3 had the weakest antimicrobial activity against BmNPV.Unlike cell level results,BmCecD1 showed synergistic enhancement of antimicrobial activity with other antimicrobial peptides in silkworm.Further statistics showed that the overexpression of antimicrobial peptides significantly delayed the larval death time after BmNPV infection between the experimental group and the control group.3.Preliminary study on antiviral mechanism of antimicrobial peptidesBmCECD1 and BmGLV3,which represent different types of antiviral peptides and have the strongest antiviral ability.In this study,the subcellular localization characteristics ofantimicrobial peptides BmCECD1 and BmGLV3 before and after infection were identified by immunofluorescence technique.The results showed that they were localized in the cytoplasm at 0-48 hours after infection.Some of them entered the nucleus 72 hours after infection.At 96 hours after infection,BmCECD1 completely entered the nucleus,while only part of BmGLV3 entered the nucleus.Because the antimicrobial peptide does not have a nuclear localization signal,we speculate that the nuclear import of BmCECD1 and BmGLV3 is related to its anti-BmNPV mechanism.Quantitative detection of BmCecD1 and BmGlv3anti-BmNPV at different time points showed that BmCecD1 and BmGlv3 had some antiviral ability from 48 h after infection.At 72-96 h after infection,the virus proliferation in the experimental group was significantly inhibited.Moreover,BmCecD1 inhibits the replication of viral genes in the late stage of infection.In order to further explore the antiviral mechanism of antimicrobial peptides,BmCECD1 and BmGLV3 were used as bait proteins to capture the interacting proteins by immunoprecipitation mass spectrometry.Immunofluorescence showed that BmNPV IE1 and BmCECD1 were co-localized in the nucleus and BmNPV DBP and BmGLV3 were co-localized in the cytoplasm.The co-precipitation experiment confirmed that BmNPV IE1 interacted with BmCECD1,BmNPV DBP and BmGLV3.Combined with the studies of BmNPV IE1 protein and BmNPV DBP protein,it is speculated that BmCECD1 may inhibit viral DNA replication by interacting with BmNPV IE1,thus inhibiting viral proliferation;BmGLV3 may inhibit viral nucleocapsid and matrix production by interacting with BmNPV DBP,thus inhibiting viral proliferation.