Function Analysis Of PCNA(proliferating Cell Nuclear Antigen) Of Autographa Californica Multiple Nucleopolyhedrovirus And Host Sf9 Cells

Proliferating cell nuclear antigen(PCNA),was initially found in the serum of patients with systemic lupus erythematosus,and was found in other species,for example,drosophila melanogaster,mammals,et al.Due to the evolutionarily conserved structure of PCNA protein,PCNA-like DNA polymerase cofactors were also found in viruses and prokaryotes.In different level of evolution,they share the same secondary structure units—a-helical andb-fold,and similar tertiary structure—a ring slip clip that slides over the DNA chain.Mammalian PCNA is an indispensable factor in the DNA synthesis process and exists in the nucleus in the form of a homotrimer.The expression level of PCNA synchronizes with the level of cell cycle.The gene AcOrf-49 is 771 bp in length.It encodes a protein with sequence homology to PCNA and has been identified in AcMNPV(Autographa california multiple nuclear polyhedrosis virus),but its role in DNA replication has not been identified.In the course of evolution,baculovirus genomes have been subjected to high level of gene loss and gene acquisition from its host.Among them,AcMNPV pcna gene(Ac-pcna)was obtained from the evolution of its host Sf9 pcna gene(Sf-pcna).In this study,we elucidated the role of Ac/Sf-pcna in AcMNPV infection process and analyzed the effect of Ac/Sf-PCNA on the expression of exogenous protein.The study consisted of the following three parts:Part?:Construction of AcMNPV-Ac/Sf-pcna-EGFP and expression analysis of Ac/Sf-PCNA-EGFP protein in the host Sf9 cells.Firstly,we used the Bac-to-Bac baculovirus recombinant expression system to construct the recombinant virus AcMNPV-Ac/Sf-pcna-EGFP by overexpressing Ac/Sf-PCNA.Then,Sf9 cells were infected by recombinant virus AcMNPV-Ac/Sf-pcna-EGFP at 5 MOI(Multiplicity of infection).Fluorescence microscopy showed that the fluorescence intensity of cells significantly increased with the prolonged infection time,and the number of infected cells also increased significantly.Western blot analysis showed that the expression of Ac/Sf-PCNA-EGFP gradually increased with the increase of infection time and reached the maximum value at 72 h p.i..Western blot for nucleoprotein indicated that expression of Ac-PCNA-EGFP could be detected at 12 h p.i.,which was 12 hours ahead of expression of Sf-PCNA-EGFP.Part?:Functional analysis of Ac/Sf-PCNA.The functional domains of Ac/Sf-PCNA were analyzed by using the GenBank and PDB databases.Their amino acid sequences were aligned by homology similarity analysis using DNAMAN software.And homologous sequence alignment showed that Ac-PCNA had 43.73%amino acid sequence homology with Sf-PCNA,but the C-terminal and N-terminal regions in these proteins were highly conservative.Firstly,Sf9 cells were infected with AcMNPV,AcMNPV-Ac/Sf-pcna-EGFP at 5 MOI for 1-3 d p.i..The copy number of genome DNA of Sf9 cells and virus was respectively quantitated by absolute quantification PCR analysis.The results showed that AcMNPV-mediated overexpression of Ac-PCNA and Sf-PCNA could stimulate replication of AcMNPV genome in the host Sf9 cells.Meanwhile,either AcMNPV-Ac-pcna-EGFP or AcMNPV-Sf-pcna-EGFP had a significant stimulating effect on Sf9 genome replication during infection.Then,Sf9cellswereinfectedwithAcMNPV-EGFPand AcMNPV-Ac/Sf-pcna-EGFP at 5 MOI.And the number of virus particles in the culture medium was detected by plaque assay,which suggested that AcMNPV-Ac/Sf-pcna-EGFP increased progeny virus yield.And the promotion of AcMNPV-Sf-pcna-EGFP on the proliferation of progeny virus wasmorethanAcMNPV-Ac-pcna-EGFP.BVproductionin AcMNPV-Ac-pcna-EGFP-and AcMNPV-Sf-pcna-EGFP-infected cells respectively was 14.53 and 19.83 fold of that in AcMNPV-infected cells at72 h p.i..Next,Sf9 cells were infected with AcMNPV-EGFP,AcMNPV-Ac/Sf-pcna-EGFP at 5 MOI for 12-48 h p.i..The results from RT-PCR analysis showed that the transcription level of ie2 gene was increased significantly during the early infection stage(before 24 h p.i.)and dropped slowly during the late infection stage(after 24 h p.i.)in the AcMNPV-Ac-pcna-EGFP treatment group,compared with that in AcMNPV-EGFP treatment group.The transcription level of 38K and vp39genes reached maximum value at 12 h p.i.in the AcMNPV-Ac-pcna-EGFP treatment group.It suggested that Ac-PCNA could dramatically improve the transcription level of ie2 gene and further improve the transcription of late gene,for example 38K and vp39.Finally,in the anti-insect activity assay,2~rdd instar larvae of Beet armyworm were fed continuously with 20?L(1×10~7pfu/mL)AcMNPV-EGFP and AcMNPV-Ac/Sf-pcna-EGFP for 5 days,respectively.The average weight,mortality,pupation and emergence rate of larvaewerecounted.Notonlythelarvaefedwith AcMNPV-Ac/Sf-pcna-EGFP grew slowly,but also the mortality rate of the larvae was higher than that in the AcMNPV-EGFP treatment group.However,the pupation and emergence rate of larvae fed with AcMNPV-Ac/Sf-pcna-EGFP were lower than that of the AcMNPV-EGFP treatment group.The average mortality rate of the larvae in the AcMNPV-Ac-pcna-EGFP and AcMNPV-Sf-pcna-EGFP treatment groups respectively was 83.33%and 91.67%,and was 1.43 and 1.57 fold of that in AcMNPV-EGFP-infected cells.The average pupation rate in the AcMNPV-Ac-pcna-EGFP and AcMNPV-Sf-pcna-EGFP treatment groups respectively was 16.67%and 8.33%,and was 0.40 and 0.20 fold of that in AcMNPV-EGFP-infected cells.The average emergence rate in the AcMNPV-Ac-pcna-EGFP and AcMNPV-Sf-pcna-EGFP treatment groups respectively was 6.67%and 3.33%,and was 0.33 and 0.17 fold of that in AcMNPV-EGFP-infected cells.These results indicated that the recombinant virus overexpressing Ac/Sf-PCNA had a strong insecticidal effect,that is,hadastronginsect-resistantactivity.Andrecombinant AcMNPV-Sf-pcna-EGFPhadhigheranti-insectactivitythan AcMNPV-Ac-pcna-EGFP.Part 3:Recombinant baculovirus AcMNPV-Ac/Sf-pcna-EGFP affected the expression of SfP53 protein,glucose metabolism pathway and the expression of exogenous protein in Sf9 cells.The first two parts of the experiments have preliminary analyzed the function of two important DNA polymerase processivity factors—Ac/Sf-PCNA,at the molecule,host cells and insect level respectively.Baculovirus can regulate the host's apoptotic pathways and metabolic pathways to facilitate its own replication throughout the infection.In this chapter,wefocusedonhowtherecombinantbaculovirus AcMNPV-Ac/Sf-pcna-EGFP affects apoptotic protein SfP53,glucose metabolism and then affects the expression of the exogenous protein Renilla Luciferase.Firstly,Sf9 cells were infected with AcMNPV-EGFP and AcMNPV-Ac/Sf-pcna-EGFP at 5 MOI from 12 to 72 h p.i..The results showed that,compared to the wild-type virus-treated group,the recombinant virus AcMNPV-Ac/Sf-pcna-EGFP delayed the expression of host cell SfP53as a whole.After the expression regularity of SfP53 in host cells was clarified,Sf9cellswerealsoinfectedwithAcMNPV-EGFPand AcMNPV-Ac/Sf-pcna-EGFP at 5 MOI,respectively.The aggregation of SfP53 in the nucleus was examined.The results showed that the aggregation of SfP53 in the nucleus was similar to that of the total SfP53 protein.Secondly,Sf9cellswereco-infectedwithAcMNPV-EGFPor AcMNPV-Ac/Sf-pcna-EGFP(0.25MOI)andAcMNPV-renilla luciferase-RFP(0.25 MOI)to detect glucose content in the host cells.The resultsshowedthatintracellularglucoseconcentrationin AcMNPV-Ac/Sf-pcna-EGFP group was significantly higher than that in AcMNPV-EGFP group at 4 h p.i..The intracellular glucose concentration in AcMNPV-Ac-pcna-EGFP-and AcMNPV-Sf-pcna-EGFP-infected cells respectively was 1.28 and 1.39 fold of that in AcMNPV-infected cells.In the following infection period,the glucose concentration decreased firstly and then increased.Furthermore,we found that the time point of glucose concentration ascension in AcMNPV-Ac/Sf-pcna-EGFP-infected cells was24-36 hours earlier than that of AcMNPV-EGFP-infected cells.And the lowest value of glucose concentration of AcMNPV-Ac/Sf-pcna-EGFP group was higher than that in AcMNPV-EGFP group.The lowest value of glucose concentration of AcMNPV-Ac-pcna-EGFP-and AcMNPV-Sf-pcna-EGFP-infected cells respectively was 1.20 and 1.37 fold of that in AcMNPV-infected cells.Finally,Sf9 cells were co-infected with AcMNPV-EGFPorAcMNPV-Ac/Sf-pcna-EGFP(0-0.5MOI)and AcMNPV-renilla luciferase-RFP(0.25 MOI)for 72 hours to detect the expression level of Renilla Luciferase.The results showed that the expression level of Renilla Luciferase in AcMNPV-Ac/Sf-pcna-EGFP treatment group decreased slowly with the increase of MOI.And,the expression level of Renilla Luciferase in AcMNPV-EGFP treatment group was significantly reduced,which indicated that AcMNPV-Ac/Sf-pcna-EGFP could weaken the down-regulation expression of exogenous proteins caused by viral infection.Sf9cellswereco-infectedwithAcMNPV-EGFPor AcMNPV-Ac/Sf-pcna-EGFP(0.25MOI)andAcMNPV-renilla luciferase-RFP(0.25 MOI)for 12 to 72 hours to detect the expression level of Renilla Luciferase.The results showed that the expression of Renilla Luciferase in AcMNPV-Ac-pcna-EGFP and AcMNPV-Sf-pcna-EGFP treatment groups respectively was detected at 48 and 24 h p.i.,which was 12and 36 hours earlier than AcMNPV-EGFP treatment group.It indicated that AcMNPV-Ac/Sf-pcna-EGFP could assist the early expression of exogenous proteins.So,AcMNPV-Ac/Sf-pcna-EGFP could delay the apoptosis of cells by delaying the expression process of SfP53,and gained more time for virus replication.At the same time,AcMNPV-Ac/Sf-pcna-EGFP stimulated the host cells to utilize glucose,which provided energy for the virus to replicate itself and was beneficial to the expression of exogenous genes.In this study,the function of Ac/Sf-PCNA involved in DNA replication of virus and host was confirmed at the DNA,host cells and insect body levels,respectively.The important role of Ac/Sf-pcna that promoted DNA replication was determined.In the meantime,AcMNPV-Ac/Sf-pcna-EGFP,two highly efficient microbial virus insecticides,were obtained and the mechanism that recombinant baculovirus AcMNPV-Ac/Sf-pcna-EGFP promoted the expression of exogenous gene by delaying the expression of apoptosis protein SfP53 and stimulating the use of glucose in Sf9 cells was evaluated.This study confirmed the function of Ac/Sf-PCNA,provided the experimental basis for the application of Ac/Sf-PCNA and laided the theoretical foundation for scientific control of lepidoptera insect.