Pseudorabies virus(PRV),an etiological agent of pseudorabies in livestock,has negatively affected the porcine industry all over the world.Epithelial cells are reported as the first site of PRV infection.However,the ro...Pseudorabies virus(PRV),an etiological agent of pseudorabies in livestock,has negatively affected the porcine industry all over the world.Epithelial cells are reported as the first site of PRV infection.However,the role of host proteins and its related signaling pathways in PRV replication is largely unclear.In this study,we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney(PK-15)epithelial cells.Totally 5723phosphopeptides,corresponding to 2180 proteins,were obtained,and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells(P<0.05).GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways.Further functional studies of NF-κB,transcription activator factor-2(ATF2),MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference(RNAi)knockdown.It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number.JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV,especially during the biogenesis of viral genome DNA.Subsequently,by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2,it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication.These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.展开更多
Activating transcription factor 2(ATF2)is a member of the ATF/cyclic AMP-responsive element bind-ing protein family of transcription factors.However,the information concerning ATF2 ion-mediated DNA binding module and ...Activating transcription factor 2(ATF2)is a member of the ATF/cyclic AMP-responsive element bind-ing protein family of transcription factors.However,the information concerning ATF2 ion-mediated DNA binding module and function of ATF2 in malignant pleural mesothelioma(MPM)has never been addressed.In this study,by using GRNInfer and GVedit based on linear pro-gramming and a decomposition procedure,with integrated analysis of the function cluster using Kappa statistics and fuzzy heuristic clustering in MPM,we identified one ATF2 ion-mediated DNA binding module involved in invasive function including ATF2 inhibition to target genes FALZ,C20orf31,NME2,PLOD2,RNF10,and RNASEH1,upstream RNF10 and PLOD2 activation to ATF2,upstream RNASEH1 and FALZ inhibition to ATF2 from 40 MPM tumors and 5 normal pleural tissues.Remarkably,our results showed that the predominant effect of ATF2 occupancy is to suppress the activation of target genes on MPM.Importantly,the ATF2 ion-mediated DNA binding module reflects‘mutual’positive and negative feedback regulation mechanism of ATF2 with up-and down-stream genes.It may be useful for developing novel prognostic markers and therapeutic targets in MPM.展开更多
基金the Natural Science Foundation of China(32170155,31770191)the Major Science and Technology Projects of Hubei Province(2021ABA005)to Z.F.L。
文摘Pseudorabies virus(PRV),an etiological agent of pseudorabies in livestock,has negatively affected the porcine industry all over the world.Epithelial cells are reported as the first site of PRV infection.However,the role of host proteins and its related signaling pathways in PRV replication is largely unclear.In this study,we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney(PK-15)epithelial cells.Totally 5723phosphopeptides,corresponding to 2180 proteins,were obtained,and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells(P<0.05).GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways.Further functional studies of NF-κB,transcription activator factor-2(ATF2),MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference(RNAi)knockdown.It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number.JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV,especially during the biogenesis of viral genome DNA.Subsequently,by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2,it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication.These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.
基金supported by the National Natural Science Foundation of China(Grant No.60673109)the Teaching and Scientific Research Foundation for the Returned Overseas Chinese Scholars,Ministry of Education of China.
文摘Activating transcription factor 2(ATF2)is a member of the ATF/cyclic AMP-responsive element bind-ing protein family of transcription factors.However,the information concerning ATF2 ion-mediated DNA binding module and function of ATF2 in malignant pleural mesothelioma(MPM)has never been addressed.In this study,by using GRNInfer and GVedit based on linear pro-gramming and a decomposition procedure,with integrated analysis of the function cluster using Kappa statistics and fuzzy heuristic clustering in MPM,we identified one ATF2 ion-mediated DNA binding module involved in invasive function including ATF2 inhibition to target genes FALZ,C20orf31,NME2,PLOD2,RNF10,and RNASEH1,upstream RNF10 and PLOD2 activation to ATF2,upstream RNASEH1 and FALZ inhibition to ATF2 from 40 MPM tumors and 5 normal pleural tissues.Remarkably,our results showed that the predominant effect of ATF2 occupancy is to suppress the activation of target genes on MPM.Importantly,the ATF2 ion-mediated DNA binding module reflects‘mutual’positive and negative feedback regulation mechanism of ATF2 with up-and down-stream genes.It may be useful for developing novel prognostic markers and therapeutic targets in MPM.
