Function Analysis Of Ac154 During The AcMNPV Infection Process In Host Cells

Ac MNPV is the first baculovirus to be sequenced and is considered a model of baculovirus.In recent years,Ac MNPV has been widely used as a biological insecticide and is called a safe insecticide.However,its low efficiency limits its wide use as an insecticide.It was proposed that a devastating pest,Spodoptera frugiperda,will enter a full-scale outbreak in China in this year.The first to be persecuted may be wheat,followed by corn and other crops.Facing such a huge food security challenge,combining various control methods and resistant control methods to reduce the overuse of pesticides is necessary.So it is urgent to find a safe and efficient biological pesticide.Therefore,in order to improve the activity of Ac MNPV as a safe insecticide,it is important to fully understand the function of genes in its genome.The last open reading frame in the Ac MNPV genome,Ac Orf-154,is 246 bp in length.So far,there is no report on its function in the virus infection process.It has been reported that bm129,the homologous gene of ac154,exists in Bm NPV.The recombinant virus obtained by knocking out this gene could increase the infectivity,but its specific function in the virus infection process is still unknown.In order to clarify the function of Ac154 in the virus infection process,we conducted a study to explore the function of Ac154.The study mainly included the following three parts:Part ?:Construction of vAc^ac154KO-EGFP,vAc^ac154KO-Ac154-EGFP and v Ac-Ac154-EGFP and expression analysis of Ac154-EGFPFirstly,we used Bac-to-Bac and ?-RED recombination systems to construct the recombinant virus vAc^ac154KO-EGFP,vAc^ac154KO-Ac154-EGFP and v Ac-Ac154-EGFP.Then Sf9 cells were infected with recombinant virus vAc^ac154KO-Ac154-EGFP and v Ac-Ac154-EGFP at 5 MOI.Fluorescence localization analysis showed that Ac154 was a protein distributed in the nucleoplasm,and was mainly distributed in the nucleus in the late stage of infection.Western blot results showed that the accumulation of Ac154-EGFP protein gradually increased with the prolonged infection time and reached the maximum level at 108 h p.i.in vAc^ac154KO-Ac154-EGFP treatment group.The accumulation of Ac154-EGFP in the nucleus was consistent with the expression of total Ac154-EGFP protein.The accumulation of total protein and nuclear protein in v Ac-Ac154-EGFP virus-infected cells showed the same trend.The expression level of Ac154-EGFP reached the top level at 72 h p.i.,and decreased at 96 h p.i..Part ?:Function analysis of Ac154 during the infection process in host cellsFirstly,TCID50 assay detected the proliferation of vAc^ac154KO-EGFP,vAc^ac154KO-Ac154-EGFP and v Ac-Ac154-EGFP progeny virus.The results showed that v Ac-Ac154-EGFP could increase the yield of progeny virus,ac154 knockout reduced the yield of progeny virus,and the replacement of ac154 could restore the progeny virus yield of vAc^ac154KO-Ac154-EGFP,which was similar to that of wild type v Ac-EGFP.Results from the previous chapter showed that the expression level of Ac154-EGFP protein continued to increase from 24 to 108 h p.i.and the expression level was still high at 108 h p.i.in vAc^ac154KO-Ac154-EGFP infected cells.It was reported that Ac MNPV-infected cells showed apoptosis after 48 h p.i..So we suspected that Ac154 might be involved in host cell apoptosis process.For this hypothesis,We measured the proliferation inhibition rate of Sf9 cells after virus infection by MTT method.The results showed that v Ac-Ac154-EGFP had a lower cell proliferation inhibition rate than v Ac-EGFP,which decreased by 5.20%,5.01% and 2.03% at 36,48,and 72 h p.i.,respectively.This result indicated that Ac154 promoted cell proliferation.P35 of Ac MNPV was the main factor to prevent apoptosis of Sf9 cells caused by viral infection according to reports,so we examined the impact of recombinant virus on transcription level of p35 gene.RT-PCR results showed that v Ac-Ac154-EGFP significantly increased the p35 transcription level,and the vAc^ac154KO-EGFP treatment could decrease the p35 transcription level compared with that in v Ac-EGFP treatment group.The results showed that Ac154 could promote the transcription of p35.In order to reveal the mechanism of Ac154 affecting cell proliferation,we finally examined the effect of recombinant virus on Sf P53 by western blotting.The results showed that vAc^ac154KO-Ac154-EGFP and v Ac-Ac154-EGFP delayed the expression of Sf P53 in host cells,compared with that in v Ac-EGFP treatment group.The above results revealed the mechanism that Ac154 delayed cell apoptosis and provided a more favorable environment for the progeny virus by regulating p35 and Sf P53,thereby promoting the proliferation of progeny viruses.Part ?:The effect of Ac MNPV p35 knockout on the function of Ac154We have clarified that Ac154 played an anti-apoptotic part during virus infection,and Ac154 promoted the transcription of p35,which mainly played an anti-apoptotic role in host cells.So what role does p35 play in the antiapoptotic effect of Ac154? Therefore,we constructed v Acp35KO-EGFP and v Acp35KO-Ac154-EGFP and analyzed the effect of v Acp35KO-EGFP and v Acp35KO-Ac154-EGFP on progeny viruses and host cells proliferation and accumulation of Sf P53 protein.TCID50 assay showed that there was no significant difference in the progeny virus production between v Acp35KO-EGFP and v Acp35KO-Ac154-EGFP treatment groups.The cell proliferation inhibition rate increased in v Acp35KO-Ac154-EGFP infected cells compared with that in v Acp35KO-EGFP treatment group during the late infection process.We detected the expression level of Sf P53 between v Acp35KO-EGFP and v Acp35KO-Ac154-EGFP treatment groups by western blotting.The results showed that there was significant difference in the expression level of Sf P53 protein between the two treatment groups during the mid-stage infection process.So,these results further demonstrated that p35 gene knockout would block the anti-apoptotic effect of Ac154 in the infected cells.p35 gene is one of the potential target genes of Ac154.In this study,the function of Ac154 in Ac MNPV infection process was confirmed at cell level,and its auxiliary role in the progeny virus proliferation was clarified.It was found that Ac154 delayed cell apoptosis by regulating the transcription of p35 and the expression of Sf P53,which gained more time for the replication of progeny virus and provided a more favorable environment to promote the generation of progeny virus.The experimental results of this research enriched the annotation function of regulatory genes in the Ac MNPV genome,provided a theoretical basis for our scientific use of Ac MNPV to control pests and to discover an efficient biological pesticide.