Study On The Regulation Mechanism Of BmNPV Iap2 Gene On Host Cell Apoptosis

Bombyx mori is an important economic insect for spinning silk as well as an significant carrier of sericulture industry and history.Bombyx mori nucleopolyhedrovirus?Bm NPV?is one of the most common and harmful species of silkworm pathogenic microorganisms in sericulture production.Bm NPV is a complete intracellular parasite.Its own genome is simple and has no cellular structure which usually uses the host's nutrients and signaling pathways to complete its life cycle.The virus infects the host cells triggers the host a series of reactions,such as apoptosis,DNA damage response,heat shock response,energy metabolism and so on.Among them,apoptosis is an important biological process of the body which is triggered by internal and external environmental changes or apoptotic signals.Apoptosis is a self-regulating way to eliminate aging,excess and damaged cells to maintain their stability under gene regulation.Apoptosis is almost present in all multicellular organisms and plays a key role in the growth and development of organisms,tissue differentiation and cellular immunity to maintain homeostasis.The virus evolves in tandem with the evolution of the host defense system,thereby escaping the antiviral mechanism of the host cell.Insects lack mammalian acquired immune system,apoptosis as a vital part of immune response,which is a excellent entry point into the study of viral and host interaction,and how the virus uses host signaling pathway to achieve its own proliferation and replication.In this study,the function of Bombyx mori nucleopolyhedrovirus apoptosis inhibitory factor Bm NPV iap2 gene was identified to explore the mechanism of Bm NPV IAP2 and its interacting protein on the apoptosis of host cells during the infection of Bm NPV.Preliminary analysis of the regulation mechanism of Bm NPV iap2 gene in Bm NPV proliferation process,which provides a theoretical basis of Bm NPV using Bm NPV iap2 to regulate host apoptosis and promote self-proliferation.The main results and conclusions are as follows:1.Functional identification of the Bm NPV iap2 geneSequence analysis revealed that the ORF of Bm NPV iap2 gene was 750 bp long and encoded 249 amino acids?aa?.The predicted protein molecular weight was 28.72 k Da and the isoelectric point was 9.51.Protein domain prediction indicated that Bm NPV IAP2 is a BIR domain from 80 aa to 153 aa,and an N-terminal RING domain from 202 aa to 236 aa.Phylogenetic tree analysis showed that the baculovirus IAP2 was mainly ?-baculovirus and had relatively high conservation.In addition,the IAP2 of baculovirus was closely related to insect IAP?especially coleoptera,Hymenoptera?.Mainly,the results are consistent with the putative baculovirus IAP through horizontal transfer of insects.The eukaryotic overexpression vector of BmNPV iap2 gene and the knockout vector based on CRISPR/Cas9 were successfully constructed.The Bm NPV iap2 gene overexpression vector was transfected into Bm E-SWU1 cells.The immunofluorescence results indicated that Bm NPV IAP2 was mainly located in the cytoplasm,and Western Blotting detected specific bands exist at 30.71 k Da;Bm NPV iap2 gene was successfully knocked out based on CRISPR/Cas9 in Bm NPV.Detection by Hoechst staining,TUNEL assay,Caspase activity assay and flow cytometry showed that in Bm NPV-infected Bm E-SWU1 cells,Bm NPV iap2 gene was knocked out,showing typical apoptotic bodies compared with the control,and the statistical results showed that the proportion of apoptotic bodies of knockout Bm NPV iap2 gene was 20% compared with the control.Compared with the control,the green fluorescence of TUNEL was significantly enhanced,the activities of Caspase-9 and Caspase-3/7 were significantly increased,and the proportion of apoptotic cells increased by 11%,indicating that Bm NPV iap2 triggered apoptosis after CRISPR/Cas9 knockout;Using q RT-PCR and Western Blotting,overexpression of Bm NPV iap2 up-regulates the expression of viral genes ie1 and vp39,promotes Bm NPV proliferation,knocks out Bm NPV iap2 down-regulates viral genes ie1 and vp39 expressed and inhibited the proliferation of Bm NPV.2.Identification of Bm NPV IAP2 interacting proteinsIn order to identify the regulatory network of Bm NPV IAP2,BmNPV IAP2 was used as a bait for co-immunoprecipitation-mass spectrometry analysis,and Bm NPV IAP2 may interact with Bm PP5 and Bm HSP60.The interaction with Bm NPV IAP2 was identified by Immunofluorescence co-localization analysis,Coimmunoprecipitation?Co-IP?assays.Bm PP5 is composed of three tandem TPR domains from 19 aa to 120 aa,and 195 aa to 471 aa is the PP2 Ac domain,which may be involved in the mitochondrial cell cycle;HSP60 is the Coiled coil domain from 359 aa to 395 aa,and the Low complexity domain from 556 aa to 572 aa,mainly involved Correct targeting of proteins to mitochondria,protein “de novo” folding,heat shock reactions,and the like.The eukaryotic overexpression vector of Bmpp5 and Bm Hsp60 gene and the knockout vector based on CRISPR/Cas9 were successfully constructed.By Hoechst staining and Caspase activity assay,Bmpp5 and Bm Hsp60 gene was knocked out,showing typical apoptotic bodies.Compared with the control,the proportion of apoptotic bodies of knockout Bm Hsp60 and Bmpp5 was 12% and 15%;in addition,the activities of Caspase-9 and Caspase-3/7 increased significantly,indicating that Bmpp5 and Bm Hsp60 triggered apoptosis after knocking out by CRISPR/Cas9.Bm HSP60 has been reported in antiviral research,this article focuses on the antiviral function of Bm PP5.By q RT-PCR and Western Blotting technology,overexpression of Bmpp5 upregulated the expression of viral genes ie1 and vp39,promotes Bm NPV proliferation;knockout Bmpp5 down-regulates the expression of viral genes ie1 and vp39,inhibits Bm NPV proliferation.At the transcriptional level and protein level,q RT-PCR and Caspase activity assays were used to further explore the regulation of Bm NPV IAP2 interacting protein on Bm NPV IAP2.The results showed that overexpression of Bmpp5 up-regulated Bm NPV iap2 and knockdown of Bmpp5 down-regulated expression of Bm NPV iap2.Caspase activity assay showed that knockdown of Bmpp5 affected Bm NPV iap2 inhibition of apoptosis.3.Bm NPV iap2,Bmiap and Bmpp5 jointly regulate host apoptosisIt is confirmed that there is an interaction between Bm NPV IAP2,Bm IAP and Bm PP5.The expression patterns of Bmiap,Bmpp5 and Bm NPV iap2 genes during Bm NPV infection were analyzed by q RT-PCR.The results showed that Bmiap expression was down-regulated and Bmpp5 and Bm NPV iap2 were up-regulated during Bm NPV infection.In order to explore the mechanism of Bm NPV hijacking Bm NPV iap2 regulation of host cell apoptosis,Bm NPV iap2,Bmiap and Bmpp5 jointly regulate host apoptosis and Bm NPV proliferation replication by q RT-PCR and Western Blotting detection means at the transcriptional and protein levels.The results showed that Bm NPV iap2 and Bmpp5 up-regulated Bmiap during Bm NPV infection.Overexpression of Bm NPV iap2 and Bmpp5 enhanced Bmiap-induced H₂O₂-induced apoptosis;in addition,overexpression of Bm NPV iap2 and Bmpp5 enhanced Bmiapinduced viral proliferation.