| Porcine parvovirus(PPV)is one of the important pathogens causing reproductive disorders in pregnant sows.The PPV genome contains two open reading frames,ORF1 and ORF2.ORF1 encodes non-structural proteins NS1 and NS2.The main function of NS1 is involved in transcriptional regulation and viral replication,and the main function of NS2 is involved in viral replication and nuclear export.It has been found that the gene encoding NS2 is contained in NS1.NS2-mRNA is formed by alternative splicing of NS1-mRNA,but its specific mechanism is still unclear.Heterogenous nuclear ribonucleoproteins(hnRNPs)are a class of multifunctional proteins that are widely expressed and highly conserved in a variety of cells.They can participate in the splicing of various viral mRNAs as splicing regulators.Therefore,members of the family may be involved in the process of PPV NS1-mRNA splicing to form PPV NS2-mRNA.In this study,hnRNPs protein was screened and identified from the host protein interacting with PPV NS1-mRNA.By interfering,knocking out and overexpression,the role of hnRNPs protein in the formation of PPV NS2-mRNA and its effect on PPV replication were clarified.The splicing site of hnRNPs protein acting on PPV NS1mRNA was further identified,and a mutant PPV strain was constructed to clarify the effect of this site mutation on PPV replication.The purpose of this study is to clarify the mechanism of PPV NS1-mRNA splicing to form PPV NS2-mRNA,and to provide a theoretical basis for further revealing the mechanism of PPV replication.The results are as follows:1.PPV NS1-mRNA interacted with host protein hnRNPK.According to the results of mass spectrometry of host proteins interacting with PPV NS1mRNA,hnRNPK,a member of the hnRNPs family,was screened to interact with PPV NS1mRNA.The eukaryotic expression vector of hnRNPK was constructed and transfected into PK-15 cells.After 24 h,the nuclear protein was extracted and incubated with biotin-labeled PPV NS1-mRNA.RNA-pulldown assay showed that PPV NS1-mRNA interacted with hnRNPK.Protein-RNA immunoprecipitation using hnRNPK antibody showed that PPV NS1mRNA interacted with hnRNPK.In PPV-infected PK-15 cells,hnRNPK and PPV NS1-mRNA were labeled with hnRNPK antibody and in situ hybridization probe,respectively.Laser confocal results showed that PPV NS1-mRNA and hnRNPK were co-localized in the cytoplasm.2.hnRNPK promotes PPV NS2-mRNA formation and PPV replication.PK-15 cells were infected with 1 MOI PPV,and the cells were collected at 0 h,12 h,24 h and 36 h.The protein and mRNA levels of hnRNPK were detected by western blotting and qPCR.The results showed that compared with the MOCK group,the protein and mRNA levels of hnRNPK were significantly increased at 24 h after PPV infection(P<0.05),and the protein and mRNA levels of hnRNPK were highly significantly increased at 36 h after infection(P<0.01).Three specific siRNAs targeting at hnRNPK were designed and synthesized,named as si-hnrnpk-270,si-hnrnpk-600 and si-hnrnpk-1090.After transfection of PK-15 cells for 36 h,qPCR and western blotting showed that si-hnrnpk-600 could highly significantly inhibit the mRNA and protein expression of hnRNPK compared with the control si-NC group(P<0.01).After transfection of PK-15 cells with si-hnrnpk-600 for 36 h,the cells were infected with 1 MOI PPV for 24 h.PCR showed that the level of PPV NS2-mRNA was highly significantly lower than that of si-NC group(P<0.01).qPCR showed that PPV DNA copy number was significantly decreased(P<0.05).The hnrnpk knockout cell line PK-15hnrnpk-/-was further constructed using the CRISPER/Cas9 system.The results of cell viability assay showed that hnrnpk knockout had no significant effect on the growth activity of PK-15 cells(P>0.05).PK-15hnrnpk-/-cells were infected with 1 MOI PPV for 24 h,and the formation of PPV NS2mRNA,PPV DNA copy number and TCID50 were detected.The results showed that compared with wild-type cells and non-knockout cells,the level of PPV NS2-mRNA in PK-15hnrnpk-/cells was highly significantly decreased(P<0.01),and the PPV DNA copy number and TCID50 were significantly decreased(P<0.01).The expression vector of NS1 and hnRNPK was co-transfected into PK-15 cells,and the protein expression and mRNA level of PPV NS1 and the mRNA level of NS2 were detected by western blotting and qPCR.The results showed that after co-transfection of hnRNPK,the protein and mRNA levels of PPV NS 1 were highly significantly decreased(P<0.01),while the mRNA level of PPV NS2 was significantly increased(P<0.05)in a concentration-dependent manner.3.hnRNPK regulates the formation of PPV NS2-mRNA by acting on the 3’end splicing site of PPV NS1-mRNA.The PPV NS1 expression vectors NS1(pC-Mut)and NS1(pN-Mut)with 3’end and 5’end splicing site mutations were constructed.PK-15 cells were co-transfected with wild-type NS1,NS1(pC-Mut)and NS1(pN-Mut)with hnRNPK for 24 h,respectively.The expression of NS1 and the formation of NS2-mRNA were detected.The results showed that compared with the wild-type NS1 group and the NS1(pN-Mut)group,overexpression of hnRNPK had no significant effect on the expression of NS1 in the NS1(pC-Mut)group(P>0.05),but the formation of NS2-mRNA was significantly reduced(P<0.05).The PPV infectious clone plasmid with PPV NS1 3’end splicing site mutation was constructed and transfected into PK15 cells.The virus was rescued by blind passage for 5 generations.PCR and sequencing confirmed that the virus was rescued successfully,named PPVpCmut.After PK-15 cells were infected with 1 MOI PPVpCmut and wild-type PPV for 24 h,the formation of PPV NS2-mRNA,PPV DNA copy number and TCID50 were detected.The results showed that compared with wild-type PPV infected cells,the NS2 mRNA level,DNA copy number and TCID50 in PPVpCmut infected cells were highly significantly decreased(P<0.01).In summary,this study screened and identified the interaction host protein hnRNPK of PPV NS 1-mRNA.Interference,knockout and co-transfected the expression vector of NS1 and hnRNPK in PK-15 cells,it was found that hnRNPK promoted PPV replication by promoting the formation of PPV NS2-mRNA.Further study found that hnRNPK acts on the 3’end splicing site of PPV NS 1-mRNA to regulate the formation of PPV NS2-mRNA,and the splicing site mutant strain PPVpCmut was constructed.It was found that the formation of NS2mRNA decreased after the splicing site mutation,and the viral replication level decreased significantly.The results clarified the mechanism by which PPV promotes NS1-mRNA splicing to form NS2-mRNA through host protein hnRNPK,laying a foundation for further revealing the mechanism of PPV replication. |
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