Function Analysis Of Ac25 During AcMNPV Infection Process In Host Cells

Baculovirus is a large,enveloped double-stranded DNA virus and infects mainly Arthropods?especially insects?.It is ubiquitous in the environment.The baculovirus that most widely studied is Autographa californica multiple nuclear polyhedrosis virus?Ac MNPV?.Although baculovirus is known as a safe biological insecticide,its application is limited due to its narrow insecticidal spectrum and low insecticidal efficiency.Researchers usually use genetic engineering methods to enhance the activity of baculovirus and improve its insecticidal efficiency.Ac MNPV orf 25 encodes a protein containing 316 amino acid residues.Ac25 protein shares high homology with DNA binding protein?DBP?in Bm NPV,which suggests that Ac25 is a potential DNA-binding protein and has similar functions to single-stranded DNA-binding protein.Previous studies showed ac25 was an essential gene because virions lacking ac25 were non-infectious and produced defective nucleocapsids.Ac25 is not a virion structural protein,it is a component in virogenic stroma,and it will not produce normal virogenic stroma after the ac25 gene is knocked out.In order to further clarify the role of Ac25 in Ac MNPV infecting host Sf9 cells,we conducted a functional study of Ac25.This research is divided into three parts:Part?: Construction of recombinant baculoviruses vAc^ac25KO-EGFP ?vAc^ac25KO-Ac25-EGFP?v Ac-Ac25-EGFP and expression analysis of Ac25-EGFPWe constructed the ac25 knockout virus vAc^ac25KO-EGFP using ?-Red homologous recombination and Bac-to-Bac expression system.After infecting Sf9 cells,we found that there was almost no green fluorescence,indicating that knockout of ac25 greatly inhibited progeny virus proliferation.In order to verify that this phenomenon was caused by the lack of ac25,we constructed the ac25 repaired virus vAc^ac25KO-Ac25-EGFP.Then,the green fluorescence level in the first,second,and third generation viruses infected Sf9 cells was gradually increased,indicating that the proliferative capacity of the progeny virus was restored after repairing ac25,and it confirmed that ac25 was crucial for the generation of progeny viruses.To further analyze the function of ac25,we constructed theover-expression virus v Ac-Ac25-EGFP.Sf9 cells were infected with vAc^ac25KO-Ac25-EGFP and v Ac-Ac25-EGFP at 5 MOI respectively,and the titer of each recombinant virus was measured.Ac25-EGFP began to be expressed at 24 h p.i.,and the expression level continued to increase from 24-72 h p.i..The expression level of Ac25-EGFP in the v Ac-Ac25-EGFP treatment group was significantly higher than that in the vAc^ac25KO-Ac25-EGFP treatment group at 24,48,60 and 72 h p.i..Part?: Functional analysis of Ac25 during Ac MNPV infection process in host cellsThe TCID₅₀ endpoint dilution method was used to detect the proliferation of the progeny viruses of vAc^ac25KO-EGFP,vAc^ac25KO-Ac25-EGFP and v Ac-Ac25-EGFP.The results showed that there was almost no progeny virus generation after ac25 was knocked out,and the progeny virus production of the repaired virus vAc^ac25KO-Ac25-EGFP was basically the same as that of virus v Ac-EGFP.However,the yield of BV after overexpression of ac25 was significantly higher than v Ac-EGFP treatment group,indicating that ac25 was necessary for the progeny virus proliferation,and overexpression of ac25 could promote the proliferation of progeny virus.Then,5 MOI v Ac-Ac25-EGFP and v Ac-EGFP was used to infect Sf9 cells for 24-96 h,and the viral genomic DNA replication level was detected by absolute quantitative PCR.The results showed that the copy number of the virus genome in the cells gradually increased from 24-96 h p.i.in v Ac-Ac25-EGFP treated cells.In v Ac-EGFP treatment group,the copy number of the virus genome also showed an upward trend,but the overall level was lower than that in the overexpression virus treatment group,indicating that overexpression of Ac25 could promote the replication of the virus genome.Then,the transcription level of the virus late genes 38 k and vp39 in the infected cells was detected by q PCR.The results showed that the transcription level of 38 k and vp39 in the v Ac-Ac25-EGFP treatment group was significantly higher than that in the v Ac-EGFP group,indicating that overexpression of Ac25 could promote the transcription of 38 k and vp39 genes.Finally,the effects of v Ac-Ac25-EGFP and v Ac-EGFP on the proliferation of Sf9 cells were examined by MTT experiments.The results showed that overexpression of Ac25 could inhibit the proliferation of Sf9 cells.Part?: Functional analysis of the nuclear localization signal sequence of Ac25In the previous chapters,we clarified the importance of Ac25 during Ac MNPV infection process,and proved that it was necessary for the generation of progeny viruses.Since Ac25 is mainly located in the nucleus,how does it enter the nucleus? In this chapter,we predicted that the amino acid residues at positions 19-23 of Ac25 protein was a typical nuclear localization signal sequence.In order to explore the function of this sequence in the entry process into the nucleus,we constructed ac25 mutant virus vAc^ac25KOAc25^{PKRER19-23AAAAA}-EGFP and v Ac-Ac25^{PKRER19-23AAAAA}-EGFP.TCID₅₀ experiment was used to test the progeny virus proliferation of these mutant recombinant viruses.Compared with the v Ac-EGFP,vAc^ac25KO-Ac25^{PKRER19-23AAAAA}-EGFP inhibited the progeny virus proliferation,and the number of progeny virus produced by the v Ac-Ac25^{PKRER19-23AAAAA}-EGFP was also significantly lower than that in the v Ac-Ac25-EGFP treatment group.5 MOI of vAc^ac25KO-Ac25-EGFP,vAc^ac25KOAc25^{PKRER19-23AAAAA}-EGFP,v Ac-Ac25-EGFP and v Ac-Ac25^{PKRER19-23AAAAA}-EGFP was used to infect Sf9 cells for 24-96 h.Western blot results showed that the accumulated abundance of Ac25-EGFP in the nucleus in vAc^ac25KO-Ac25^{PKRER19-23AAAAA}-EGFP treatment group was significantly lower than that in vAc^ac25KO-Ac25-EGFP treatment group.In short,the mutation of Ac25 nuclear localization signal sequence reduced the accumulation of Ac25-EGFP in the nucleus of infected cells,which led to the reduction of the progeny virus production.This study revealed the function of Ac25 in the process of Ac MNPV infecting host cells.It relied on its nuclear localization signal to highly accumulate in the nucleus,and promoted the replication of viral DNA and the expression of late genes,thereby stimulated the generation of progeny viruses.These results further improved the annotation of the genome function of Ac MNPV,and provided experimental data and theoretical basis for analyzing the mechanism of action of Ac MNPV on host cells and developing microbial pesticides with high anti-insect activity.