Effects And Mechanism Of Host Proteins SYNCRIP And MCM3 On The Replication Process Of Porcine Parvovirus

Porcine parvovirus(PPV)is the major pathogen causing reproductive failure in sows and has caused huge economic losses to the pig industry,and characterized by abortion,stillbirth,deformed and mummified fetuses in primiparous sows.The PPV genome contains two open reading frames O RF1 and ORF2.ORF1 mainly encodes non-structural proteins NS1 and NS2,and its function is involved in virus replication and cytopathy.NS2 has the same N-terminus as NS1 and is generated by NS1-m RNA(pre-m RNA)undergoing alternative splicing.The host protein Syncrip is a member of the heterogeneous nuclear ribonucleo protein(hn RNPs)family,which is involved in the process of host m RNA transcription,exon splicing and selection of splice sites.In addition,Syncrip could also participate in the replication of some hepatitis viruses.ORF2 mainly encodes structural proteins VP1 and VP2.VP2 is the most important capsid protein in the PPV,accounting for about 80% of the capsid components.In the process of virus infection,VP2 could participate in the entry of the viral genome into the nucleusand the assembly of mature virus particles.The host protein MCM3 is a member of the helicase MCM family,which can unwind the double-stranded DNA during host DNA replication and promote the replication of host cell DNA.However,the roles and mechanisms of host protein Syncrip and MCM3 on PPV replication have not been reported so far.In the present study,an E.coli expression vector encoding NS1 and NS2 the common gene sequence was first constructed,and polyclonal antibodies that can both recognize NS1 and NS2 were prepared.The In-Fusion cloning technology was used to construct a full-length infectious clone of PPV that stably carried genetic markers.Then,the protein-protein Co-Immunoprecipitation(Co-IP)and RNA-protein immuno-precipitation(RNA-pulldown)tandem mass spectrometry were used to screened the key host proteins that interact with NS1-m RNA and VP2,Syncrip and MCM3,respectively.Finally,the roles and mechanisms of the interacting host proteins Syncrip and MCM3 in the process of PPV replication we re studied,which laying a theoretical foundation for further research on the pathogenesis of PPV.The results are as follows.1.The ns gene shared by NS1 and NS2 amplified by PC R was cloned into the prokaryotic expression vector p ET-32 a.The recombinant prokaryotic expression vector p ET-32a-NS was constructed,and identified by PCR,restriction enzyme digestion and sequencing,and then was transformed into E.coli Rosetta(DE3)for induced expression.The results of SDS-PAGE and western blotting showed that the recombinant plasmid expressed an NS recombinant protein of approximately 35 k Da in both supernatant and inclusion body.After optimizing the optimal conditions of soluble expression of recombinant bacteria,it was concluded that the expression was the highest at 37 ? with 0.8 m M inducer concentration for 6 hours.Under the optimal conditions,a large number of recombinant proteins were purified by His nickel column.Anti-NS protein serum antibod y was prepared by immunizing mice with the purified recombinant protein.The titer of the antibody was 1:12800 detected by ELISA.The result of western blotting showed that the antibody could specifically recognize the recombinant protein NS expressed in prokaryotic cells and the NS1 and NS2 expressed in eukaryotic cells.2.The special palindrome sequences F1 and F2 at both ends of the genome were artificially synthesized to construct the plasmid p KQLL(F1+F2)containing the F1 and F2 sequences.Using the extracted PPV DNA as a template,F3 and F4 fragments were amplified respectively,and In-fusion cloned with the linearized plasmid p KQLL(F1+F2)to construct a full-length PPV infectious clone plasmid p Y-PPV carrying a genetic marker.The plasmid p Y-PPV was transfected into PK-15 cells,and the virus was recued by successive passages.The DNA of infected cells was extracted and tested positive by viral nucleic acid PCR.Viral particles were observed under electron microscopy,and the expression of virus capsid protein was detected by western blotting and immunofluorescence(IFA),and the genetic markers was still detectable in successive passages to the 15 th generation.The above results indicated that the PPV infectious clone that stably carried the genetic marker was successfully rescued and named as Y-PPV strain.The Y-PPV and the parental strain PPV strains was inoculated with PK-15 cells at the same MOI,and replication capacity of above two strains was tested by measuring the virus DNA copy number and TCID50 at different time points after infection.At the same time,the apoptosis induced by PPV and Y-PPV strains were detected by AO-EB staining,flow cytometry and caspase activity.The results showed that the replication ability of Y-PPV was not significantly different from that of the parent strain(p >0.05),and it had similar apoptosis-inducing properties as the parent strain.Furthermore,the PPV antibody level in serum of primiparous sows in Y-PPV and PPV infected groups was consistent and no significant difference at each time point(p >0.05)by ELISA.The results of immuno-radiometric detection showed that the progesterone levels in the serum of the Y-PPV and PPV-infected groups had the same trend and no significant difference between the two groups at each time point(p >0.05).There was no significant difference of viral loading in the same tissue infected by Y-PPV and PPV by Real-time PCR(p >0.05).Histopathological examination results showed that both Y-PPV and PPV infections could cause obvious pathological damage to uterine and ovarian tissues.3.To screen the key interacting host proteins of NS1-m RNA.Biotin-labeled NS1-m RNA was synthesized in vitro,and it was co-incubated with nuclear extracts of PK-15 cells.Co-precipitated proteins were screened with streptavidin magnetic beads and analyzed by mass spectrometry to identify key hosts that interact with NS1-m RNA.The biotin-labeled NS1-m RNA and PK-15 cells nuclear extracts were co-incubated,and the results of RNA-pulldown showed that there was interaction between NS1-m RNA and Syncrip.Protein-RN A immuno-precipitation which performed by co-incubation with Syncrip antibody and nuclear extract of PK-15 cells infected PPV showed that NS1-m RNA interacted with syncrip.In situ hybridization was performed after PK-15 cells were infected with PPV.The results of laser confocal detection showed that NS1-m RNA and Syncrip were co-localized.The biotin-labeled NS1-m RNA and purified prokaryotic Syncrip were tested for interaction in vitro,and the results of gel migration showed that there was a direct interaction between NS1-m RNA and Syncrip.4.To clarify the role and mechanism of Syncrip in PPV infection.The copy number of virus DNA and TCID50 in the supernatant of PPV infected cells which treated with syncrip si RN A were measured,and the results showed that interference with the expression of Syncrip could reduce the copy number of DNA and TCID50 during PPV infection(p <0.05).The syncrip knockout cells was successfully constructed by CRISPR-Cas9 gene editing technology.The test results showed that the cell viability of this cell line was not significantly different from that of wild type PK-15 cells(p >0.05).In syncrip knockout PK-15 cells infected with PPV,the absence of Syncrip could significantly reduce DNA copy number and TCID50(p <0.05),and significantly reduce the protein expression level and m RN A level of NS2(p <0.05).The ns2 deletion strain was constructed using Y-PPV as the operating platform,and the cell test results showed that ns2 deletion can significantly reduce the virus DNA copy number a nd TCID50 during PPV infection(p <0.05).The Real-time PCR and western blotting were used to detect the positive correlation between the replication level of PPV in tissues and the expression level of Syncrip.When ns1 and syncrip were co-transfected with in vitro,western blotting and Real-time PC R results showed that over-expression of Syncrip could significantly reduce the protein and m RNA expression level of NS1(p <0.05).When the Syncrip co-transfected with Flag-NS1(p C-Mut)with a splice site mutation at the 3'end of ns2 on ns1,western blotting results showed that over-expression of Syncrip had no significant effect on the protein expression of NS1(p >0.05).5.To screen and identify the key interacting host proteins of VP2.The protein of PK-15 cells infected with PPV was collected,and co-immunoprecipitation and mass spectrometry were performed with PPV capsid protein 3C9 antibody to screen a key host protein MCM3 that interacting with VP2.PCI-His-VP2 and p CI-Flag-MCM3 were co-transformed into HEK293 T cells,and the interaction between VP2 and MCM3 was found by Co-IP.The immuno-fluorescence staining of PK-15 cells co-transfected with p CI-His-VP2 and p CI-Flag-MCM3 plasmids or virus infected showed the co-localization of exogenous and endogenous VP2 and MCM3 by the laser confocal microscopy.Furthermore,p ET-32a-VP2 and p GEX-4T were expressed in E.coli and purified to detect the interaction between VP2 and MCM3 by GST-pulldown and His-pulldown,which was independent of PPV infection or other molecule s.6.To clarify the role and mechanism of VP2 and MCM3 in PPV replication.The copy number of virus DNA and TCID50 in the supernatant of PPV infected cellswhich treated with VP2 si RN A were measured,and the results showed that interference with the expression of VP2 could significantly reduce copy number of virus DNA and TCID50 after PPV infection(p <0.05).There was no significant difference in cell viability between the mcm3 knockout cell line successfully constructed by CRISPR-Cas9 gene editing technology and the wild type PK-15 cells(p >0.05).The cell infection test showed that the loss of mcm3 could significantly reduce DNA copy number and TC ID50(p <0.05).It was identified that 115 aa~231 aa of VP2 was the key domain interacting with MCM3 by Co-IP.It was further observed by in fluorescent situ hybridization that NS1,VP2 and MCM3 interacted with viral genomic DNA after PPV infection by the laser confocal microscop y.The direct interaction between VP2,NS1 and viral genomic DNA was detected by DNA pulldown,while MCM3 could not directly interact with viral genomic DNA.Co-IP and laser confocal detection found that NS1 could not participate in recruiting MCM3 to viral genomic DNA,while VP2 could directly recruit MCM3 to viral genomic DNA.It was further detected that 300 nt~356 nt of genomic DNA was the key motif region interacting with VP2 by DNA-pulldown and mutation of this interaction motif could completely prevent the replication of PPV.In this study,a polyclonal antibody with high specificity and capable of recognizing both PPV non-structural proteins NS1 and NS2 was prepared.The PPV infectious clone named Y-PPV was successfully obtained with genetic stability,which could be distinguished from the parental strain,and retained the biological characteristics similar to that of the parental strain.Syncrip and MCM3,key host proteins that interact ing with NS1-m RNA and VP2,were screened,respectively,and the direct interactions between NS1-m RNA and VP2 and the key host proteins Syncrip and MCM3 were clarified.It was found that NS2 and Syncrip could participate in the process of PPV replication.K nockout of syncrip significantly reduced the replication of PPV and the expression of NS2,and the deletion of ns2 also significantly reduced the replication capacity of PPV.The over-expression of Syncrip could reduce the expression of NS1,and this site of action was located at the 3'end splice site of ns2 on ns1.VP2 and its interacting protein MCM3 were involved in PPV replication.During PPV infection,VP2 and viral genomic DNA directly interacted to recruit MCM3 to the viral genome DNA to promote PPV replication.It was found that VP2(115 aa~231aa)was the key domain interacting with MCM3 and the 300 nt~356 nt of viral genome was a key motif for viral genomic DNA to interact with VP2,which was necessary for PPV replication.The above results clarified the role and mechanism of Syncrip and MCM3,the interaction proteins of NS1-m RNA and VP2,in the process of PPV replication,which provide a theoret ical basis for further study on the pathogenesis of PPV.