Effect And Mechanism Of SUMOylation Of Host Protein NPM1 On PCV2 Replication

Porcine circovirus type 2（PCV2）is an important pathogen that harms the pigs industry.The severity of the disease caused by PCV2 is related to its replication level in vivo.It’s reported that Cap protein is indispensable for PCV2 replication,and Cap can regulate the PCV2 replication process by interacting with various host proteins.Previous studies found that in PCV2-infected wild-type PK-15 cells,the expression level of nucleophosmin 1（NPM1）,which interacts with Cap,did not change significantly with virus replication,but in the npm1 gene knockout PK-15 cells,the replication level of PCV2 was significantly reduced.However,it is reported that NPM1 usually exerts its corresponding functions through its own different post-translational modifications.Therefore,we speculate that in PCV2-infected PK-15 cells,NPM1 may be involved in the regulation of PCV2 replication through its post-translational modifications.However,what kind of post-translational modification of NPM1 that regulates PCV2 replication and its specific mechanism remains unclear.This study intends to confirm the post-translational modification mode and specific post-translational modification sites of NPM1 in PCV2-infected PK-15 cells,exploring the specific molecular mechanism of PCV2-induced post-translational modification of NPM1.At last,the effects of post-translational modification on interaction with Cap and specific replication stages of PCV2 were found out which laying atheoretical data for further studies of PCV2 replication mechanism.The results are as followed:1.PCV2 infection promoted SUMOylation of the host protein pNPM1The results showed that compared with the MOCK group,the expression level of NPM1（38 k Da）in the PCV2-infected group did not change significantly（P>0.05）,but a band higher than 250 k Da was detected,which increased significantly from 6 h to 18 h of infection（P<0.01）,and decreased to the same level in MOCK group at 24 h of infection（P>0.05）.Using si RNA to interfere with the npm1 gene in PK-15 cells,it was found that compared with the transfection control（si-NC）group,the level of NPM1（38 k Da）and NPM1（higher than 250 k Da）in si RNA#1 and si RNA#2 groups significantly reduced（P<0.01）.In npm1 knockout PK-15(PK-15^npm1-/-)cells,neither NPM1（38 k Da）nor NPM1（higher than 250 k Da）protein bands were detected regardless of PCV2 infection.The comparison found that porcine NPM1（pNPM1）contains two SUMOylation sites（K230 and K263）identical to human NPM1（h NPM1）,which can significantly increase the molecular weight of the modified protein.The pNPM1 SUMOylation modification site mutation vectors pNPM1（K230R）,pNPM1（K263R）and pNPM1（K230R&K263R）were constructed and transfected into PK-15^npm1-/-cells respectively.After PCV2 infection,the results of western blotting showed that,compared with the wild-type pNPM1 group,the level of pNPM1（higher than 250 k Da）in the cells of the pNPM1（K230R）group was decreased,but not significantly（P>0.05）,and the level of pNPM1（higher than 250 k Da）in the cells of the pNPM1（K263R）group Extremely significant reduction（P<0.01）,pNPM1（higher than 250 k Da）was not detected in cells in the pNPM1（K230R&K263R）group.2.The SUMOylation of pNPM1 was caused by the activation of the ERK/SAE1/Ubc9 pathway by PCV2 CapSUMO1,SUMO2 and SUMO3 eukaryotic expression vectors were constructed,and co-transfected into PK-15^npm1-/-with wild-type pNPM1 expression vector respectively,1MOI PCV2 infected cells for 12 h.Laser confocal results showed that in MOCK cells,SUMO1,SUMO2,SUMO3 were present in the cytoplasm and nucleus,and co-localized with pNPM1 in the nucleolus;in PCV2-infected cells,SUMO1,SUMO2 and SUMO3 were present in the cytoplasm and nucleus,while pNPM1 was almost exclusively localized in the nucleolus,and the three SUMOs were obviously co-localized with pNPM1 in the nucleolus.The results of Co-IP assay showed that PCV2 infection promoted the interaction of NPM1with SUMO2 and SUMO3（P<0.01）.After 1 MOI PCV2 infected PK-15 cells for different times,western blotting showed that compared with MOCK cells,the expression level of SAE1 did not change significantly with the prolongation of PCV2 infection time（P>0.05）,while the expression level of Ubc9 was significantly up-regulated at 6h-18 h after PCV2infection（P<0.01）.and there was no significant change at 24 h（P>0.05）.PK-15 cells were infected with recombinant adenovirus expressing PCV2 Rep（r Ad-Rep）,Cap（r Ad-Cap）,ORF3（r Ad-ORF3）and blank recombinant adenovirus（r Ad-blank）,respectively.western blotting showed that compared with r Ad-blank,only r Ad-Cap infection significantly upregulated SUMOylated pNPM1 levels（P<0.01）.PK-15 cells were treated with different signaling pathway inhibitors and infected by PCV2 for 12 h.Western blotting results showed that compared with DMSO-treated cells,the expression level of SUMOylated pNPM1 in ERK inhibitor-treated cells was significantly down-regulated（P<0.01）,while there were no significant changes in other inhibitor-treated cells（P>0.05）,At the same time,the expression level of Ubc9 was also significantly decreased（P<0.01）.3.SUMOylation of pNPM1 promoted PCV2 replicationThe wild-type and different SUMOylation site mutant pNPM1 vectors were co-transfected with GFP-Cap expression vector into PK-15^npm1-/-cells respectively.Laser confocal results showed that wild-type pNPM1 and pNPM1（K230R）co-localized with Cap in the nucleolus,pNPM1（K263R）and Cap only co-localized in a small amount in the nucleolus,while pNPM1（K230R&K263R）did not co-localize with Cap.Co-IP results showed that compared with the wild-type pNPM1 group,the interaction between pNPM1（K230R）and Cap was not significantly changed（P>0.05）,the interaction between pNPM1（K263R）and Cap was significantly reduced（P<0.01）,while pNPM1（K230R&K263R）did not interact significantly with Cap.Wild-type pNPM1 and different SUMOylation point mutants were transfected into PK-15^npm1-/-cells respectively,and infected with PCV2 at 1 MOI for 12 h,and the PCV2 TCID₅₀ and DNA copies number were determined respectively.The results showed that pNPM1（K230R）had no significant effect on PCV2 replication compared with wild-type pNPM1（P>0.05）,while both pNPM1（K263R）and pNPM1（K230R&K263R）mutants significantly inhibited PCV2replication（P<0.01）.The results of in situ hybridization assay showed that compared with the wild-type pNPM1 group,the PCV2 template and replication chain in the pNPM1（K230R）group did not change significantly（P>0.05）,while the PCV2 template and replication chain in the pNPM1（K263R）and pNPM1（K230R&63R）groups was significantly reduced（P<0.01）.In this study,it was found for the first time that PCV2 infection can promote the SUMOylation of pNPM1.It is cleared that the ERK/SAE1/Ubc9 pathway was activated by Cap to promote the SUMOylation of pNPM1 after PCV2 infection of PK-15 cells,and it is found that the SUMOylation of pNPM1 promoted its interaction with Cap to promote PCV2 replication.The findings lay the foundation for the further revealing of the PCV2replication mechanism.