| The genomes of most viruses are relatively simple,so the encoded protein is relatively small.The successful infection of the virus with the organism usually depends on the material and function of the host cell.In the different infection stages of the virus,the normal physiological structure and function of the host cell are usually changed,which is used for the replication and propagation of the virus itself.Baculovirus is the largest group of known insect viruses,and it is also one of the earliest discovered,most studied,and meaningful insect viruses.Bombyx mori nucleopolyhedrovirus,a representative of baculovirus,is also the pathogen of the important disease of Bombyx mori.Therefore,taking BmNPV as the research object,explaining the mechanism of BmNPV regulating host cell is not only of great biological significance to improve the mechanism of baculovirus,the main group of pathogens,regulating host replication and proliferation,but also can provide theoretical support for the prevention and control of Bombyx mori main pathogen BmNPV.In previous studies,we found that BmNPV can induce cell cycle arrest in the G2/M phase and promote self-proliferation and replication,but the key viral factors and host factors that affect cell cycle arrest are still unclear.In view of this,this study first screened and determined that BmNPV lef-11 can arrest cell cycle in G2/M phase.Furthermore,the mechanism of BmNPV LEF-11 regulating Bombyx mori induced metalloproteinase inhibitor protein-like was determined.The characteristics of BmIMPI expression and BmIMPI regulation of host cell cycle were analyzed regulation of host cell cycle were analyzed.Meanwhile,the mechanism by which BmNPV LEF-11 mediates BmIMPI to regulate the host cell cycle is determined.This study elucidates the mechanism by which the interaction between BmNPV LEF-11and the host protein BmIMPI affects the cell cycle progression of Bombyx mori,and improves the study of the interaction mechanism between baculovirus and host cells,which can provide a theoretical basis for the prevention and control of BmNPV.The main results are as follows:1.Regulation of BmNPV lef-11 on cell cycleTo identify the effect of BmNPV lef-11 on the cell cycle,the changes in cell proliferation were detected after over-expression of BmNPV lef-11,and the results showed that the cell proliferation activity decreased compared with the control.The effect of BmNPV lef-11 on DNA replication was detected by Ed U-labeling.The results showed that at 24 h,48 h and 72 h after over-expression of BmNPV lef-11,compared with the control,the Ed U-labeled cells were significantly reduced,indicating that cell DNA replication was inhibited.Flow cytometry analysis of cell cycle changes showed that over-expression of BmNPV lef-11 arrested the cell cycle in the G2/M phase,and the cells in the G2/M phase increased significantly.To explore the regulatory mechanism,this study detected the changes in cell proliferation and cell cycle-related genes under the condition of over-expression of BmNPV lef-11.The results showed that the expression levels of proliferating cell nuclear antigen,G2/M checkpoint cyclin B and cyclin dependent kinase 1 were down-regulated compared with the control after BmNPV lef-11 over-expression.Western blotting analysis results showed that BmCyclin B protein expression level was decreased compared with the control after over-expression of BmNPV LEF-11.Co-IP verified whether there was a direct interaction between BmNPV LEF-11 and BmCyclin B,and the results showed that there was no direct interaction between BmNPV LEF-11 and BmCyclin B.The above results indicate that BmNPV lef-11 can regulate host cell cycle changes by arresting the cell cycle in G2/M phase and down-regulating the expression level of cyclins such as BmCyclin B.However,there is no direct interaction between BmNPV LEF-11 and BmCyclin B,and the mechanism of how BmNPV LEF-11 regulates cell changes is still not understood.Therefore,the interaction protein of BmNPV LEF-11 in cells is captured to explore the mechanism of its regulation of cell cycle.2.Analysis of basic characteristics of BmIMPI and verification of interaction with BmNPV LEF-11Previous studies have found that BmNPV lef-11 can regulate cell cycle.To study the mechanism of its regulation of cell cycle,BmNPV LEF-11 interacting proteins were obtained by co-immunoprecipitation and yeast two-hybrid.Based on the analysis of gene function,BmIMPI was screened from the candidate proteins of BmNPV LEF-11 interaction,and its cloning and basic characteristics were analyzed.Sequence analysis showed that BmIMPI is rich in a large number of cysteine residues,and it is predicted to have two trypsin inhibitor like cysteine rich domains,suggesting that it may have protease inhibitory activity.Homologous sequence alignment and phylogenetic tree found that BmIMPI has low sequence conservation in other species.Tissue expression profile analysis showed that BmIMPI was highly expressed in silk glands,and period expression profile analysis showed that BmIMPI expression was lower in dormancy,higher expression in full feeding period,and higher in 5th instar larvae to migratory period.It is speculated that BmIMPI is involved in cell proliferation and cell cycle.To verify the interaction between BmIMPI and BmNPV LEF-11,the cells were transfected with p IZ-LEF11Flag and p IZ-IMPIHA-EGFP plasmids for 48 hours and then BmNPV was added for induction.Immunofluorescence co-localization found that BmIMPI and BmNPV LEF-11 could be co-localized in the nucleus.Therefore,it is speculated that BmIMPI and BmNPV LEF-11 may have interaction.Furthermore,the interaction between LEF-11 and BmIMPI was verified by immunoprecipitation technology and Western bloting technology,and it was found that BmIMPI and BmNPV LEF-11 had an interaction.The results of q RT-PCR showed that the expression of BmIMPI increased after BmNPV lef-11 over-expression compared with control.The above results indicate that BmNPV LEF-11 interacts with BmIMPI,thereby regulating the expression of BmIMPI.3.Explore the regulation of BmIMPI on the cell cycleBased on gene function analysis,to determine that BmNPV lef-11 regulates BmIMPI and affects the cell cycle,this study detected the changes in cell proliferation after over-expression of BmIMPI in BmN-SWU1.The results showed that compared with the control,cell proliferation activity decreased after over-expression of BmIMPI.The Ed U results showed that compared with the control,the cells labeled with Ed U were significantly reduced after over-expression of BmIMPI,that is,cellular DNA replication was inhibited,resulting in inhibition of cell proliferation.Flow cytometry analysis found that over-expression of BmIMPI arrested the cell cycle in the G2/M phase and increased cells in the G2/M phase.These results suggest that BmIMPI regulates cell proliferation by arresting cell cycle in G2/M phase.To verify that BmIMPI is a key gene that regulates the cell cycle in cells,this study detected the effect on cell proliferation after interfering with the expression of BmIMPI.The results showed that the cell proliferation activity increased after intervention of BmIMPI,and the number of Ed U-labeled cells increased significantly,indicating that the replication of cell DNA was promoted.Flow cytometry analysis showed that G2/M phase cells decreased after BmIMPI interference.The above results indicate that BmIMPI is involved in cell cycle regulation.4.Study on the mechanism of BmNPV lef-11 regulating host cell cycle through BmIMPITo verify that BmNPV lef-11 regulates the host cell cycle through BmIMPI,BmNPV lef-11 was over-expressed and BmIMPI was down-regulated in this study.Ed U results showed that when BmNPV lef-11 was over-expression while interfering with BmIMPI,there was no significant difference in the number of labeled cells compared with the control,indicating that BmNPV lef-11 regulates host cell DNA replication through BmIMPI.Flow cytometry analysis of the cycle changes proved that after over-expressing BmNPV lef-11 while interfering with BmIMPI,there was no significant difference in G2/M phase cells.The above results prove that BmNPV lef-11 regulates host cell proliferation through BmIMPI.To explore the mechanism of BMIMPI regulation of cell cycle,this study found that after over-expression of BmIMPI,compared with the control group,the expression levels of BmPCNA、BmCyclin B and BmCDK1 were significantly down regulated at 24 h after over-expression of BmIMPI.At 36 h after BmIMPI over-expression,the expression levels of BmCyclin B and BmCDK1 were significantly down regulated.Compared with the control,the expression level of BmPCNA was up-regulated at 24 h after BmIMPI intervention,and the expression of BmCyclin B and BmCDK1 were up-regulated at 36 h after BmIMPI intervention.Western blotting analysis showed that the expression of BmCyclin B protein was significantly decreased after over-expression of BmIMPI,while the expression of BmCyclin B protein was significantly increased after interference with BmIMPI.Immunofluorescence showed that BmIMPI could co-localize with BmCyclin B.Therefore,this study identified that BmNPV LEF-11 interacts with BmIMPI and regulates the cell cycle by regulating the expression of downstream genes,thereby inhibiting host cell proliferation. |
