Function Analysis Of Ac132 In AcMNPV During The Infection Process In Host Cells

Baculoviruse is a class of double-stranded,circular,supercoiled DNA viruses that contain a genome from 80 kb to 180 kb and encode approximately 100-180 proteins.Baculovirus has been widely studied,especially Autographa californica multiple nuclear polyhedrosis virus?Ac MNPV?.Although Ac MNPV has the title of biosafety insecticide,it is also not widely applied due to its various limitations,including slow speed of lethality and so on.Therefore,it is particularly important to continuously improve the toxicity of baculovirus and fully understand the function of key genes,especially the structural gene.ac132 is located in the 132 th open reading frame of the Ac MNPV genome.ac132 gene is 660 bp in length and encodes an important structural protein.Previous research proved that knockout of ac132 had no significant effect on viral DNA replication and expression of late genes vp39 and e25.However,knockout of ac132 had an effect on the morphogenesis of the virus.When ac132 was knockout,the virogenic stroma?VS region?had almost no rod-like nucleocapsid,but a curly hair-like structure appears and the virion contained only a single nucleocapsid,and delay of the virogenic stroma formation was observed.However,the effect of Ac132 on the proliferation of progeny virus was still not clear,so we explored the function of Ac132 in the infection process in host cells.This study is mainly divided into three parts:Part?Construction of AcMNPV^ac132ko-EGFP,AcMNPV^ac132ko-Ac132-EGFP and Ac MNPV-Ac132-EGFP and expression analysis of Ac132-EGFPFirstly,we constructed AcMNPV^ac132ko-EGFP harboring EGFP tag by using lambda-Red homologous recombination and bac-to-bac system.In the green fluorescence maps of the first,second and third generation virus in host cells,low expression of green fluorescent protein indicated that ac132 gene knockout significantly reduced the generation of Ac MNPV progeny virus.In order to determine whether this phenomenon is indeed caused by the knockout of ac132 gene,we constructed AcMNPV^ac132ko-Ac132-EGFP.A large amount of green fluorescence was observed in the green fluorescence map of the first,second and third generation viruses,and the preliminary observation by fluorescence microscope indicated that ac132 gene was indispensable.In order to further research on ac132 gene,we constructed Ac MNPV-Ac132-EGFP.To analyze the expression of Ac132-EGFP,5 MOI AcMNPV^ac132ko-Ac132-EGFP and Ac MNPV-Ac132-EGFP was used to infect Sf9 cells.Western blot analysis showed that the expression of Ac132-EGFP in the AcMNPV^ac132ko-Ac132-EGFP treatment group started at 24 h p.i.and almost reached the peak level at 36 h p.i..In the Ac MNPV-Ac132-EGFP treatment group,the expression of Ac132-EGFP started at 12 h p.i.,which was earlier than that in the AcMNPV^ac132ko-Ac132-EGFP treatment group,and reached the high expression peak at 36 h p.i.,and it showed high expression abundance in the subsequent infection period.The results from fluorescence microscopy observation were consistent with those from Western blot analysis.Part ? Function of Ac132 during the infection process in host cellsFirstly,plaque assay detected the proliferation of AcMNPV^ac132ko-EGFP,AcMNPV^ac132ko-Ac132-EGFP and Ac MNPV-Ac132-EGFP progeny virus.The results showed that the ability of produced progeny virus by AcMNPV^ac132ko-Ac132-EGFP was similar to that of wild type Ac MNPVEGFP.However,Ac MNPV-Ac132-EGFP significantly promoted the proliferation of progeny viruses,which was 5.55 times of that in Ac MNPV-EGFP group and 2.28 times of that in AcMNPV^ac132ko-Ac132-EGFP group at 36 h p.i.,respectively.So how does the overexpression of Ac132 promote Ac MNPV to produce progeny viruses? Previous research had shown Ac132 was structural protein of Ac MNPV and it was localized in the nucleus.We analyzed accumulation of Ac132 in the nucleus in AcMNPV^ac132ko-Ac132-EGFP and Ac MNPV-Ac132-EGFP infected Sf9 cells and the nucleoproteins.The results showed that Ac132 was highly accumulated in the nucleus in both virus treatment groups.So Ac132 plays a role in the nucleus,but how does it get into the nucleus? Therefore,c NLS Mapper,an online prediction website,preliminarily predicted that there was a typical nuclear localization signal sequence PPKK?proline-proline-lysinelysine?at 25-28 amino acid residues of Ac132 protein sequence.In order to clarify whether the PPKK sequence plays a role in the entry process into the nucleus,we mutated PPKK into alanine and construct AcMNPV^ac132koAc132^{PPKK25-28AAAA}-EGFP and Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP.The progeny virus proliferation of these two mutant viruses was determined by plaque assay.The results showed that,compared with the wild type Ac MNPV-EGFP,the generation of the progeny virus of AcMNPV^ac132koAc132^{PPKK25-28AAAA}-EGFP treatment group significantly reduced.Compared with wild type,the production of Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP progeny virus at 48 h p.i.was almost 4 times of that in Ac MNPV-EGFP group,but it was still lower than that in the Ac MNPV-Ac132-EGFP group.The fluorescence maps of various recombinant virus transfected or infected Sf9 cells were correspond with their ability to produce progeny virus,which indicated the importance of nuclear localization signal of Ac132.These above results suggested that Ac132 played a role by entering the nucleus with nuclear localization signal,and leading to a large number of progeny viruses.In addition,studies had also shown that the 103-134 amino acid residues of Ac132 sequence were homologous with the NEBU domain,an actin binding domain.Actin played an important role in the whole process of Ac MNPV infection in the host and generation of progeny virus.Then,how does Ac132 overexpression or mutation affect F-actin? 5 MOI recombinant viruses AcMNPV^ac132ko-Ac132-EGFP,Ac MNPV-Ac132-EGFP,AcMNPV^ac132ko-Ac132^{PPKK25-28AAAA}-EGFP and Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP was used to infect Sf9 cells at 12-72 h p.i..F-actin was stained with phalloidine and distribution of F-actin in the nucleus was observed by fluorescence microscopy.In Ac MNPV-Ac132-EGFP treatment group,F-actin aggregated obviously in the nucleus at 12 h p.i.,and lasted until 72 h p.i.and a mass of F-actin also accumulated in the inner side of the cell,which aggregated significantly earlier than other treatment groups.In the AcMNPV^ac132koAc132^{PPKK25-28AAAA}-EGFP treatment group,F-actin almost concentrated in the intracellular side at 12-48 h p.i.,and appeared in the nucleus at 72 h p.i..Actin distribution in the nucleus and cytoplasm of AcMNPV^ac132ko-Ac132-EGFP treatment group and Ac MNPV-EGFP was similar.They were gradually entering the nucleus at 12-48 h p.i.,and began to depolymerate after 72 h p.i..Compared with the Ac MNPV-EGFP treatment group,F-actin in the Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP treatment group entered the nucleus in advance and delayed exnucleus,but it was still inferior to the Ac MNPV-Ac132-EGFP treatment group.Then we detected the aggregation of beta-actin in the nucleus by Western blot,and these results were consistent with the results from fluorescence localization analysis.The above results indicated that Ac132 entered the nucleus by its nuclear localization signal on an incomplete reliance manner,and could affect actin aggregation in the nucleus in advance,then promote the proliferation of Ac MNPV.Part ? Effects of AcMNPV^ac132ko-EGFP,AcMNPV^ac132ko-Ac132-EGFP,Ac MNPV-Ac132-EGFP,AcMNPV^ac132ko-Ac132^{PPKK25-28AAAA}-EGFP and Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP on energy metabolism in host cellsAc MNPV proliferation can activate PI3K-Akt-m TOR signaling pathway,and the signaling pathways can activate the downstream signaling pathways associated with cell metabolism.Akt regulates cellular process of glycolysis,and PI3K-Akt signaling pathway may activate pyruvate kinase of the glycolysis process and raise the level of glycolysis.So how does the recombinant virus affect glycolysis in Sf9 cells? Glucose consumption and lactic acid accumulation can reflect the level of glycolysis.5 MOI recombinant viruses AcMNPV^ac132ko-EGFP,AcMNPV^ac132ko-Ac132-EGFP,Ac MNPV-Ac132-EGFP,AcMNPV^ac132ko-Ac132^{PPKK25-28AAAA}-EGFP and Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP was used to infect Sf9 cells for 6-72 h p.i.,and the consumption of glucose and the accumulation of lactic acid in Sf9 cells were detected.The results showed that the stronger the proliferative ability of progeny virus,the more glucose consumption and the more lactic acid accumulation in Sf9 cells.In the Ac MNPV-Ac132-EGFP treatment group and the Ac MNPV-Ac132^{PPKK25-28AAAA}-EGFP treatment group,Sf9 cells showed high glucose consumption at 12 h p.i.,which was about 1.82 times and 1.18 times of that in Ac MNPV-EGFP treatment group,respectively.At the same time,a large amount of lactic acid was also accumulated at 6 h p.i.,about 2.66 times and 1.42 times of that in the Ac MNPV-EGFP treatment group,respectively,but the increase of glucose consumption was slowed down afterwards.In the AcMNPV^ac132ko-Ac132-EGFP treatment group and the Ac MNPV-EGFP treatment group,the glucose uptake of Sf9 cells and the accumulation of lactic acid were generally consistent,and the glucose demand was significantly increased at 24 h p.i.,and then showed a slow increase.In the AcMNPV^ac132ko-EGFP and control treatment group,the glucose consumption of Sf9 cells continued to increase slowly,with almost no accumulation of lactic acid.In the AcMNPV^ac132ko-Ac132PPKK25-28 AAAAEGFP treatment group,Sf9 cells began to increase glucose uptake at 24 h p.i.due to slow accumulation of progeny viruses,and the accumulation of lactic acid also increased slowly,until a large amount of lactic acid suddenly appeared at 72 h p.i.,which may be caused by the high accumulation of progeny viruses at 72 h p.i..In conclusion,the recombinant virus harboring Ac132 expression or mutant promoted or inhibited the proliferation of progeny virus,and then affected the glycolysis process in Sf9 host cells.It also provided a clue to further clarify the energy metabolism of the key structural genes of Ac MNPV in host cells.This study revealed the important function of Ac MNPV structural protein Ac132 by constructing a recombinant virus harboring knockout,overexpression and mutation of ac132 at the cellular and molecular level,it relied on the autonuclear localization signal to accumulate in the host cell nucleus,and polymerized in the nucleus through the early fibrous actin,and promoted progeny virus proliferation and activated cellular energy metabolism in host cells.It accelerated the glycolysis process of cells to provide energy for virus replication,assembly and release.These results provide a reference for the scientific use of baculovirus in biological control of plant pests.