Effect Of Activating Transcription Factor ATF2 On PRV Proliferation In PK-15 Cells

Pseudorabies virus?PRV?is a pathogen belonging to alpha herpesvirus family which causes huge economic losses in the pig industry by inducing Pseudorabies every year.It is necessary to study PRV proliferation activities and the interaction between PRV and host cell factors for finding the treatment drug targets of Alpha-herpes virus and development of new vaccines.In this study,we regard pseudorabies virus?PRV?as a model of alpha herpesviruses.To obtain a systems-level overview of signaling events in PK-15 cells during PRV infection,we conducted phosphoproteomics profiling of a PRV-infected PK-15 cells by iTRAQ combined with LC-MS/MS technology.And then we knockdown gene expression by siRNA,inhibit MAPK signal pathway?ERK,JNK,p38?conduction,construct atf2-null cell line base on lentivirus Cas9 expression system,overexpress complete atf2 and phosphorylation site mutant atf2?T69A,T71A?for assessing role of atf2 gene and MAPK Pathway in PRV proliferation.It is expected to further probe the proliferation mechanism of PRV in epithelial cells from the perspective of host quantitative phosphoproteomic,which provide a valuable resource for searching PRV proliferation-dependent host genes,for exploring potential therapeutic targets,and for developing the new vaccines.The work contents and research results involved in this study are as follows:1.Quantitative phosphoproteomic analysis of PK-15 cell by iTRAQThe PK-15 cells were infected by PRV at MOI of 10,and after 2.5 hpi,the cells were lysed.Here,we performed isobaric tags for relative and absolute quantitation?iTRAQ?quantitative phosphoproteomics on PRV-infected PK-15 cells,which identified 5723 phospho-peptides,corresponding to 2180 protein including 810 proteins that significantly changed in phosphorylation level at 2.5 h of exposure to PRV according to screening criteria of Ratio>+/-1.2 and P value<0.05,implying that the PRV infection has extremely complex effects on host protein phosphorylation.KEGG differential enrichment analysis showed that PRV infection influences MAPK signaling pathway including several kinases and cellular factors.2.Validation of phosphoproteomic data and preliminary screening of atf2 geneTo further confirm the phosphoprotemics data rationality,we took the phosphospecific antibodies to validate the iTRAQ phosphorylation protein ratios.we chose atf2(pT⁶⁹/pT⁷¹)and other ribosomal kinase rsk2(pS³⁷⁸)from the phosphoprotemics.Immunoblot result assessed that the ATF2 and RSK2phosphorylation levels were enhanced upon the addition of PRV for 2.5 h,which were consistent with the states of phosphoprotemics identification.To address whether PRV-responsive phosphoproteins in MAPK pathway are important for PRV replication,we selected nf?b,atf2,max,sos genes which locate in the different subpathways of MAPK pathway and have changed in phosphorylation level with PRV infection according to MAPK pathway map.The results showed that the PRV titers of the control group were 10^7.15 PFU/mL,and when atf2 gene was down-regulated,the PRV titers were 10⁶.55.55 PFU/mL and down-regulated by 10^0.6PFU/mL.When rsk2 gene was down-regulated,PRV titers were 10^6.35 PFU/mL and down-regulated by 10⁰.8.8 PFU/mL,suggesting that knockdown of atf2 or rsk2 reduced PRV copy number and proliferation.3.Effect of inhibitor blocking MAPK Pathway signaling on PRV replicationIn order to determine MAPK Pathway that may participate in PRV replication activities,we used inhibitors U0126,SP600125 and SB202190 for ERK,JNK and p38 signaling pathways.Western blotting assess blocking effect of the inhibitors,showing that U0126 significantly down-regulated the phosphorylation level of RSK2and had a better inhibitory effect on ERK Pathway signaling.For JNK and p38signaling pathways involved in ATF2,the results suggested that only SP600125played a role in down-regulating the phosphorylation level of ATF2 through the JNK Pathway,PRV activate atf2 mainly through the JNK Pathway.To explore the mechanism by which PRV elicits the activation of JNK signaling pathway,we treated PK-15 cells with WT-PRV or UV irradiated-inactivated PRV and evaluated ATF2 phosphorylation by Western blotting.UV irradiated-inactivated PRV did not affect ATF2 phosphorylation at Thr69,Thr71 compared with uninfected-PRV,suggesting that active infectious virus is required to stimulate JNK pathway and ATF2phosphorylationFinally.In the summary,plaque assay results indicated that PRV replication on PK-15depended on JNK Pathway phosphorylation and activation of atf2 gene.Inhibition of JNK Pathway signaling inhibited the replication of PRV on PK-15 cells by the inhibitor SP600125 down-regulated ATF2 phosphorylation level.4.Effect of atf2 gene knockout on PRV proliferation in PK-15 cellsOn the basis of constructing the atf2-deletion Cas9 PK-15 cell line,we use the MOI=0.05 of WT-PRV to infect ATF2-/-1,ATF2-/-4 Cas9 PK-15 cells,and monitored PRV titers of three time point by plaque assay,12h.p.i.,PRV titers in WT-PK15 were 10^8.45 PFU/mL;in atf2-null cell lines,PRV titers were 10^7.68 PFU/mL and 10⁷.69.69 PFU/mL,down-regulated by 10^0.77 PFU/mL and 10^0.76PFU/mL,indicating that PRV proliferation have been significantly suppressed in atf2-null cell lines.5.Effect of overexpression of atf2/atf2?T69A,T71A?gene on proliferation of PRV in PK-15 cellsFurthermore,in order to figure out the effect of ATF2 protein or the translated phosphorylation of ATF2 alone on virus replication,we individually overexpressed complete ATF2 and T^69/71?T to A?amino acid locus mutant expression plasmids in PK-15 cells.The results of plaque assay showed that the PRV titers of the control group pcDNA3.1-FLAG group were 10^5.59 PFU/mL,the PRV titers of the pc pcDNA3.1-FLAG-ATF2group were 10^5.68 PFU/mL,and the PRV titers of the pc pcDNA3.1-FLAG-mATF2?T69A,T71A?group were 10^5.57 PFU/mL.This result effectively demonstrated that the ATF2 phosphorylation plays a pivotal role in PRV replication.