Role And Mechanism Of Host Cell Proteins STAT5,npm1 And DNAJB6 In The Process Of PCV2 Replication

Porcine circovirus type 2?PCV2?is the primary pathogen causing porcine circovirus-associated diseases?PCVDs?.PCV2 infection invades the immune system,not only causing infected pigs to produce immunosuppression and become susceptible to a variety of pathogens,but also resulting a significant increase in the fatality rate of infected pigs and a significant decline in the production performance,and leading to huge economic losses to the pig industry.The PCV2 genome contains 11 overlapping open reading frames?ORFs?,while the most important of which is ORF2.ORF2 encodes a capsid?Cap?protein which identified as a major viral structural protein.Cap participates in the entry of the virus genome into the nucleus and the assembly of mature virus particles in the process of PCV2 replication.Signal transducer and activator of transcription5?STAT5?is a multifunctional transcription factor with DNA binding properties.Nucleolar protein nucleophosmin?NPM1?is a multifunctional host protein that not only participates in a variety of cellular processes,but also plays an important role in the replication of multiple viruses.Heat shock protein40 B6?DNAJB6?is a member of the heat shock protein 40 family and is involved in the folding and transport of protein in cells,the assembly of protein complexes,the degradation of misfolded proteins and the regulation of virus replication.However,the roles and mechanisms of STAT5,NPM1 and DNAJB6 in the process of PCV2 replication are still unclear.In this study,the host cell proteins STAT5,NPM1 and DNAJB6 that interacting with Cap were screened by immunoprecipitation.Further,the roles and mechanisms of STAT5,NPM1 and DNAJB6 in the process of PCV2 replication were studied respectively,which laying a theoretical foundation for further exploration of the pathogenic mechanism of PCV2.The results are as follows.1.The Cap antibody was used for immunoprecipitation,and the key host interaction proteins STAT5,NPM1 and DNAJB6 of Cap were screened.The primers for stat5,npm1 and dnajb6 genes were designed for PCR to amplify the porcine stat5,npm1 and dnajb6 genes and cloned into the eukaryotic expression vector p CI-neo.The results of restriction digestion and sequencing showed that p CI-His-STAT5,p CI-Flag-NPM1 and p CI-Flag-DNAJB6 plasmids were successfully constructed.The results of sequence comparison with the same protein of human and murine sequences show that the amino acid sequence of porcine STAT5 and murine STAT5 had the highest homology?96.3%?.The amino acid sequence of porcine NPM1 has the highest homology?98.2%?with human NPM1.1 MOI PCV2 was infected with PK-15.Indirect immunofluorescence staining revealed that Cap co-localized with STAT5 and NPM1localized in the nucleus,and co-localized with DNAJB6 in the nucleus and cytoplasm.The culling of pigs 28 days after PCV2 infection found that STAT5,NPM1 and DNAJB6 can be detected in the heart,liver,spleen,lung,kidney and lymph nodes of pigs.The protein and m RNA levels of NPM1 and STAT5 were not significantly different from those in control tissues?p>0.05?,while the protein and m RNA level of DNAJB6 in livers,lungs,kidneys and lymph nodes was significantly higher than that in the control tissues?p<0.05?.2.The plasmids of p EGFP-Cap and p CI-His-STAT5 were co-transfected into HEK293T for immunoprecipitation,and the results showed that Cap can binds to STAT5.GST-pull down detection of the interaction between Cap and STAT5 found that Cap cannot directly interacted with STAT5.PCV2 DNA copies in the si RNA#1 group with the highest interference efficiency was significantly lower than that in the non-specific si RNA transfection group?p<0.05?.PCV2 DNA sequence contains a GAS-like motif?5'TTCTCTGAA3'?,which is a conserved binding site for STAT5 protein.DNA pull down confirmed that PCV2 GAS-like motif mutant infectious clones?PCV2-MDS?cannot bind to STAT5,while wild-type PCV2 DNA can bind to STAT5.After PK-15 infected with equal dose of wild-type PCV2 or PCV2-MDS for 12 h and 24 h,the replication of PCV2 DNA was detected by FISH.Comparing with wild-type PCV2-infected cells,the number of probes?RFP?targeting the viral genome DNA replicational chain?negative strand?and probes?CP?targeting the viral genome template strand?positive strand?were significantly reduced in PCV2-MDS infected cells at 24 hpi?p<0.05?;Fluorescence quantitative PCR detection showed that PCV2 DNA copies of PCV2MDS infected cells was significantly lower than that in wild-type PCV2 infected cells at 12 hpi and24 h hpi?p<0.05?.3.PK-15,which interfered with npm1 gene,was infected with PCV2 for 24 h.It was found that the number of viral DNA copies in the cells transfected with npm1 si RNA#1significantly decreased compared with the control cells?p<0.05?.The CRISPR/Cas 9technology was used to construct npm1 knockout PK-15,the number of viral DNA copies in npm1 knockout PK-15 cells was significantly reduced compared with wild-type PK-15?p>0.05?.GST-pull down results showed that STAT5 can directly interact with NPM1,and NPM1 can directly interact with Cap.Construct NPM1 truncated plasmids and Cap truncated eukaryotic expression plasmids,the binding areas of NPM1?151-158 aa?and Cap?136-153 aa?were determined by GST-pull down.After PK-15 infected with PCV2 mutant strain?PCV2-Nm A?,which mutated 147 arginine and 148 histidine of Cap to alanine,viral DNA copies in PCV2-Nm A infected cells was significantly decreased compared with these in wild-type PCV2 infected cells?p<0.05?.The results of fluorescence in situ hybridization showed that the number of RFP and CP positive cells in the PCV2-Nm A infected cells was significantly lower than those in the wild-type PCV2 infected cells?p<0.05?.4.PK-15 was infected with 1 MOI PCV2.Compared with control group,the DNAJB6m RNA level significantly increased at 36 hpi?p<0.05?,and further increased at 48 hpi?p<0.01?.The change of p DNAJB6 m RNA level was consistent with the change of p DNAJB6protein level.Then PK-15 were infected with different doses of PCV2 for 36 h,and the results showed that the expression of DNAJB6 increased with PCV2 infection dose.Co-immunoprecipitation showed that DNAJB6 interact with Cap,and further GST-pull down determined that DNAJB6 can directly interact with Cap.The binding regions of DNAJB6 and Cap were determined to be 1-99 aa of DNAJB6 and 162-234 aa of Cap.Wild-type PK-15,dnajb6 knockout PK-15(PK^DNAJB6-/-)and control cells(PK^DNAJB6+/+)were infected with same dose of PCV2,respectively.The results showed that the level of Cap protein and progeny virion production in PK^DNAJB6-/-were significantly lower than that in wild-type PK-15 and PK^DNAJB6+/+?p<0.05?.The number of autophagosomes induced by PCV2 infection or Cap expression in PK^DNAJB6-/-was significantly lower than that in wild-type PK-15 and PK^DNAJB6+/+?p<0.01?.Wild-type PK-15,dnajb6 overexpressing PK-15(PK^DNAJB6)and blank vector transfected PK-15?PK^V?were infected with same dose of PCV2,Cap protein expression and progeny virion production significantly increased in the PK^DNAJB6 relative to PK-15 and PK^V?p<0.05?,and the number of autophagosomes in PK^DNAJB6 significantly increased compared with wild-type PK-15 and PK^V?p<0.05?.Plasmids p CI-Flag-DNAJB6?J and p EGFP-Cap?162-234 were constructed.The wild-type PK-15 stably expressing GFP-LC3 was transfected into p CI-neo,p CI-Cap or p CI-Cap?162-234 for 24 h.The results showed that compared with cells transfected with p CI-Cap,the number of autophagosomes in p CI-Cap?162-234 transfected cells was significantly reduced?p<0.01?.Transfected p CI-p DNAJB6,p CI-p DNAJB6?J and p CI-neo plasmids in stably expressing GFP-LC3 PK^DNAJB6-/-,and then infected with same dose of PCV2 or transfected same dose of p CI-Cap plasmid.The results showed that the number of autophagosomes in p CI-p DNAJB6?J transfected cells was significantly lower than that in p CI-DNAJB6 transfected cells?p<0.01?,and the number of viral DNA copies in the p CI-p DNAJB6?J transfected cells was significantly reduced compared with the p CI-p DNAJB6 transfected cells?p<0.01?.PK^DNAJB6-/-was treated with autophagy inhibitor 3-MA or DMSO,and then respectively transfected with p CI-p DNAJB6,p CI-p DNAJB6?J and p CI-neo,finally infected with same dose of PCV2.In PK^DNAJB6-/-transfected with p CI-DNAJB6,the level of virus production in 3-MA treatment decreased significantly compared with DMSO treatment?p<0.01?.PK^DNAJB6 was pretreated with 3-MA/DMSO and then infected with same dose of PCV2,and the results showed that the level of virus production in 3-MA treatment cells was significantly lower than in DMSO treatment cells?p<0.01?.PK^DNAJB6 was treated with atg5 si RNA and then infected with PCV2,and the results showed that inhibiting the expression of atg5 significantly reduced the formation of autophagosomes and the level of virus production in PCV2-infected cells.In this study,the host cell proteins STAT5,NPM1 and DNAJB6 that interact with PCV2Cap were screen.STAT5 indirectly interacted with Cap and can combined with the GAS-like motif of PCV2 DNA,and obtained a mutant strain PCV2 with a lower replication ability than wild-type PCV2.It was found that NPM1 can directly interact with Cap and STAT5,and the interaction region between NPM1 and Cap was identified,and the mutant strain at Cap 147and 148 mutation strain was obtained.The replication ability of this strain was significantly lower than that of wild-type PCV2.PCV2 infection up-regulates the expression of DNAJB6,and Cap 162-234 aa directly binds to DNAJB6 1-99 aa.Mutant DNAJB6/Cap interaction sites,treatment with autophagy inhibitors,or inhibition of atg5 gene expression can significantly reduce the level of autophagosome and virus production induced by PCV2 infection.The above results clarified the roles and mechanisms of host cell proteins STAT5,NPM1 and DNAJB6in the process of PCV2 replication,and provided a theoretical basis for further revealing the pathogenic mechanism of PCV2.