Function Analysis Of Inhibitors Of Apoptosis Ac-IAP1/2 Of AcMNPV

Inhibitors of apoptosis,IAPs,were initially found as a kind of anti-apoptotic proteins in baculoviruses,and were found in other species later,such as mammals,drosophila melanogaster,nematodes,et al.In baculoviruses,IAPs are a large class of proteins that regulate the process of host cell apoptosis and are now divided into six classes according to the homology of their amino acid sequences,IAP1-6.The structures of various types of IAPs are similar,but their functions are different and some IAPs promote the apoptosis of their host cells.The host cells carry out antiviral action by initiating its apoptotic procedure,and the virus promotes its own proliferation by inhibiting apoptosis.In order to clarify the function of Ac-IAP1/2 in the insect resistance mechanism of Autographa california multiple nuclear polyhedrosis virus(AcMNPV),the functional study of Ac-IAPs were performed in this study,and on this basis,the synergistic effects of Ac-IAP1/2 on apoptisis activity of recombinant AcMNPV-BmK IT(P10)were investigated.The study was divided into three parts:Part I: Functional analysis of inhibitors of apoptosis Ac-IAP1/2Firstly,we constructed the recombinant virus Ac MNPV-iap1/2-egfp by Bac-to-Bac baculovirus recombinant expression system to over express Ac-IAP1/2 in host cells.Then,Sf9 cells were infected with AcMNPV-iap1/2-egfp at 1 MOI(Multiplicity of infection),and the results from fluorescence microscopy detection showed the expression level of Ac-IAP1/2-EGFP increased with the prolongation of infection time and reached the maximum at 120 h,which were consistent with these from western blot analysis.Subsequently,Sf9 cells were infected with AcMNPV and AcMNPV-iap1/2-egfp at a concentration of 0.001 MOI,and plaque assay was used to detect the number of virus particles in the cells and culture medium of these treatment groups,which suggested that AcMNPV-iap2-egfp promoted progeny virus multiplication,whereas AcMNPV-iap1-egfp inhibited progeny virus multiplication,which demonstrated that Ac-IAP2 promoted progeny virus multiplication and Ac-IAP1 proformed the opposite function.Following,Sf9 cells were infected with AcMNPV and AcMNPV-iap1/2-egfp at a concentration of 1.5-12 MOI for 48 h,respectively,and the proliferation of Sf9 cells was detected by MTT reagent.The results showed AcMNPV-iap2-egfp could promote cell proliferation in a dose-dependent manner while AcMNPV-iap1-egfp significantly inhibited cell proliferation under normal or nutritional deficiencies stress condition,which suggested that Ac-IAP1 induced cell apoptosis and Ac-IAP2 promoted cell proliferation.In the anti-insect activity assay,third instar helicoverpa armigera larvae were fed continuously with 20 ?L(1 × 107 pfu/m L)AcMNPV-egfp and AcMNPV-iap1/2-egfp for 5 days,respectively.The results of the growth,mortality and pupation rate of larvae showed that the larvae fed by AcMNPV-iap2-egfp grew slowly,and its mortality was also higher than the other two treatment groups.However,the mortality and pupation rate of larvae fed by AcMNPV-iap1-egfp was almost the same with these in AcMNPV-egfp group.That is,although the cytotoxicity of Ac-IAP1 was strong,the AcMNPV-iap1-egfp,which over expressed Ac-IAP1,had poor insecticidal activity,but AcMNPV-iap2-egfp could be used as a microbial insecticide with high anti-insect activity.Part II: BIR2 domain was essential for the anti-apoptotic function of Ac-IAP2GenBank and PDB database were used to search the functional domains of Ac-IAP1/2,their domains were analyzed by homology similarity analysis using DNAMAN software,and the difference domain-BIR2 was determined.Then,the BIR2 domain of Ac-IAP1/2 was replaced by overlapping PCR and the recombinant virus AcMNPV-iap2-BIR2(iap1)-egfp,which could over express the mutant Ac-IAP2-BIR2(iap1),was constructed by the Bac-to-Bac system.Then,Sf9 cells were infected with AcMNPV-iap2-BIR2(iap1)-egfp(1 MOI),the results from fluorescence microscopy and western blot analysis showed that the number of Sf9 cells harboring green fluorescence and intracellular green fluorescence intensity was significantly enhanced with the prolongation of infection time.The expression level of Ac-IAP2-BIR2(iap1)-EGFP increased and reached the top at 120 h p.i.(hours post-infection).Subsequently,Sf9 cells were infected with AcMNPV-iap1/2-egfp and AcMNPV-iap2-BIR2(iap1)-egfp at the concentration ranged from 3 to12 MOI for 48 h respectively,and the proliferation rate of Sf9 cells was detected by MTT reagent.The results showed that AcMNPV-iap2-BIR2(iap1)-egfp,differed from AcMNPV-iap2-egfp by only the BIR2 domain,had higher inhibition rate than that in AcMNPV-iap1-egfp group,which suggested that Ac-IAP2-BIR2(iap1)promoted apoptosis and the BIR2 domain was essential for the anti-apoptotic function of Ac-IAP2.Part 3: Synergistic effect analysis of Ac-IAP1/2 on pro-apoptotic activity of recombinant virus AcMNPV-BmK IT(P10).The first two parts of the experiments confirmed the function of Ac-IAP1/2,two important regulation factors in host cell apoptosis.Buthus martensii Karsch(BmK)is a kind of scorpions which venom contains excitatory insect toxins(Bm K IT).In previous work,we engineered BmK IT gene into the genome of AcMNPV and BmK IT was inserted to the downstream of late(P10)and very late(Polh,PH)promoters,respectively.Whether or how they would carry out the synergistic effect on the cytotoxicity of the recombinant virus AcMNPV-BmK IT(P10)? In view of previous studies,BmK IT,which was regulated and expressed by the promoter of IE1,could significantly promote progeny virus multiplication and Sf9 cell apoptosis in the early stages of viral infection.And BmK IT,which regulated by early and late promoters(IE1/P10/PH),could induce apoptosis of Sf9 host cells.We first analyzed the effect of BmK IT,which was regulated and expressed by P10 promoter,on progeny virus multiplication.Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(P10)at 1 MOI,respectively.Viral titer was used to quantify the of progeny virus in infected cells.It showed that the accumulation of virus particles in AcMNPV-BmK IT(P10)treated cells was lower than that in wild virus treatment group within 16-48 h p.i.,that is,AcMNPV-BmK IT(P10)inhibited the accumulation of progeny virus in host cells.Then,Sf9 cells were infected with AcMNPV-BmK IT(P10)at 1 MOI,the results from Real-time PCR showed that the transcriptional level of BmK IT was weakened after 24 h p.i..And the number of progeny virions in the host cells was slightly increased at 48 h p.i.,which suggested that BmK IT was indeed the cause of the accumulation of progeny virus particles in host cells.Following,Sf9 cells were infected with AcMNPV-BmK IT(P10)at 1 MOI.The Real-time PCR analysis showed that the transcription level of vp39 in AcMNPV-BmK IT(P10)treated group was higher than that in wild virus treatment group in the early stage of viral infection,but it was lower in the late stage of viral infection.It was presumed that BmK IT,which was regulated by the P10 promoter,promoted progeny virus multiplication in the early stage,and inhibited the proliferation of progeny virus in the late stage in the viral infection process.Combined with the results from virus titer test,we concluded that the BmK IT,which was regulated by P10 promoter,promoted progeny virus multiplication and budding in the early stage virus infection,but it promoted cell apoptosis and inhibited multiplication of progeny virus to promote transmission in the late stages of infection.Subsequently,Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(P10)at 10 MOI,respectively.The results from DAPI fluorescent staining showed there was no significant change in the cell nucleus morphology in each virus-treated group,which suggested the effect of recombinant virus AcMNPV-BmK IT(P10)on the multiplication and budding of progeny virus did not depend on the formation of the virogenic stroma.After clarifying the pattern of multiplication of AcMNPV-BmK IT(P10)in the host cells,we examined the synergistic effect of Ac-IAP1/2 on the pro-apoptotic activity of AcMNPV-BmK IT(P10).Sf9 cells were co-infected with AcMNPV-iap1/2-egfp(3 MOI)and Ac MNPV-BmK IT(P10)(1.5,3,6 MOI),respectively.The results from MTT assay showed Ac-IAP1 could promote host cell apoptosis in collaboration with BmK IT,but its cytotoxicity was much higher than that of Bm K IT;Ac-IAP2 provided a delayed time for the infection of AcMNPV-BmK IT(P10)by inhibiting apoptosis,which could promote the proliferation of progeny viruses and increased the cytotoxicity of AcMNPV-BmK IT(P10).In this study,the function of Ac-IAP1/2 was confirmed at the insect and host cells level respectively and the important role of the BIR2 domain in the anti-apoptotic function of Ac-IAP2 was determined.At the same time,a highly efficient microbial virus insecticide AcMNPV-iap2-egfp was constructed and the synergistic effect of Ac-IAP1/2 on the pro-apoptotic activity of AcMNPV-BmK IT(P10)was evaluated.These results provided a theoretical and experimental basis for the mechanism and application of baculovirus IAPs.And it is significance for improving the insecticide activity of insect baculovirus and scientific control of lepidoptera insect.