The Mechanism Of Porcine Parvovirus VP2 Promote Own Replication By Hijacking Host Protein MCM3

Porcine parvovirus（PPV）is a single negative stranded DNA virus with autonomously replicating.The replication of PPV depends on the replicase system of host cells completely.The PPV genome mainly contains two open reading frames（ORFs）,ORF1 and ORF2.ORF1 encodes non-structural proteins NS1,NS2 and NS3;O RF2 encodes structural proteins VP1 and VP2.VP2 is the most important structural protein of PPV,accounting for about 90%of the viral capsid components.And it also has immunogenicity,which leads to the immune response.VP2 plays a significant part in the process of viral replication.Minichromosome maintenance protein 3（MCM3）is a subunit of the MCM2～7 complex.It regulates the assembly of the MCM complex and binds to the DN A replication origins of cell to affect the replication of cellular DNA.It reported that the MCM complex could regulate the replication process of various viruses,such as adeno-associated virus,herpes virus,human parvovirus.We have found that VP2 interacted with MCM3 to recruit it to the viral replication origins to promote the replication of the virus during the process of PPV infection in the previous research.However,the mechanism of how VP2 hijacks MCM3 to promote viral replication,is still unclear.In this study,polyclonal antibodies against VP2 or NS1 were prepared respectively.Then,real-time PCR was used to detect the replication level of virus and cellular DNA after PPV infection in PK-15 cells,and the Co-IP and Ch IP were used to detect the ability of MCM3 binding to histone H3 and DNA replication origins of cells after PPV infection in PK-15 cells to study the impact of PPV infection on host cellular DNA replication and viral replication.Finally,the expression and phosphorylation level of MCM3were detected after PPV infection in PK-15 cells to determine the phosphorylation site of MCM3 and the key viral proteins that could regulate MCM3 phosphorylation;the effect of MCM3 phosphorylation on VP2-MCM3 interaction,MCM3-histone H3 interaction and PPV replication were detected.The research was aimed to elucidate the mechanism of VP2hijacking MCM3 to promote viral replication.It provides a theoretical basis for further exploration of the pathogenic mechanism of PPV.The results of the study are as follows.1.PPV virions were amplified and purified.The prokaryotic expression vector p ET-32a-ns1s,a specific fragment of NS1（1395 nt～1986 nt）,was constructed,and the recombinant protein of NS1S was obtained after expression and purification.BA LB/c mice were immunized with the purified PPV virions and NS1S recombinant protein respectively,and mouse polyclonal antibodies against VP2 or NS1 of PPV were successfully prepared respectively.The results of ELISA and western blotting showed that the titers of mouse anti-VP2 and that of anti-NS1 polyclonal antibodies were both 1:256000,and they could well recognize the prokaryotic/eukaryotic expression of VP2 or NS1 recombinant protein and endogenous VP2 or NS1 of PPV after PPV infection in PK-15 cells.2.PK-15 cells were infected with PPV at 1 MOI for 0,12,and 24 h post infection,and the level of PPV and DNA replications were detected by real-time PCR.The results showed that the copies of PPV virus increased significantly with the infection time prolonged（P<0.05）,and DNA copies of the host cells were significantly reduced（P<0.05）.Co-IP and Ch IP were used to detect the binding of MCM3 to histone H3 and MCM3 to the DNA replication origins of cells,Lamin b2 prom and dhfr prom,after PPV infection.The results of Co-IP showed that,compared with Mock control group,the ability of MCM3 binding to histone H3 was significantly reduced（P<0.05）in the PPV infected group;The results of Ch IP showed that,compared with the Mock control group,the ability of MCM3 binding to the DNA replication origins of cells,Lamin b2 prom and dhfr prom,was markedly reduced（P<0.05）in the PPV infected group.3.PK-15 cells were infected with PPV at 1 MOI for 0,12,and 24 h post infection,and the expression and phosphorylation level of MCM3 were detected by western blotting.The results showed that the expression of total protein of MCM3 was insignificant differences（P>0.05）,but the level of phosphorylation of the 160th threonine of MCM3 was significantly reduced（P<0.05）with the infection time prolonged.The recombinant plasmids,pCI-Flag-ns1、pCI-His-vp2,were transfected into PK-15 cells,then,the expression and phosphorylation level of MCM3 were detected by western blotting at 0,12,24,36 h after transfection.The results showed that the phosphorylation level of the 160th threonine of MCM3 was significantly reduced（P<0.05）in NS1 recombinant plasmid transfected group,while the phosphorylation level of the 160th threonine of MCM3 was not significantly changed（P>0.05）in VP2 recombinant plasmid transfected group with the transfection time prolonged.Recombinant plasmid,pCI-His-vp2,was co-transfected with pCI-neo or pCI-Flag-ns1 into PK-15 cells.The results of Co-IP showed that,compared with the pCI-His-vp2 and pCI-neo co-transfected group,the ability of MCM3 binding to VP2 was increased significantly（P<0.05）in the pCI-Flag-ns1 and pCI-His-vp2 co-transfected group.Recombinant plasmid,pCI-Flag-ns1,was transfected into PK-15 cells.The results of Co-IP showed that,compared with the pCI-neo transfected group,the ability of MCM3 binding to histone H3 was increased significantly（P<0.05）in the pCI-Flag-ns1 transfected group.At last,cell strains of Flag-MCM3^T160A overexpressed were constructed.Wild-type PK-15 cells,PK-15^MCM3 cells and PK-15^MCM3T160A cells were infected with PPV at 1 MOI,then,the virions copies,TCID₅₀ and the expression of VP2 of PPV were detected at 24 h post infection.The results showed that,compared with wild-type PK-15 cells infected with PPV group,the virions copies and TCID₅₀ were significantly increased（P<0.05）in the PK-15^MCM3 and PK-15^MCM3T160A cells infected with PPV groups.Meanwhile,the expression of VP2 of PPV was also significantly increased（P<0.05）;compared with PK-15^MCM3 cells infected with PPV group,the virions copies and TCID₅₀ were significantly increased（P<0.05）in PK-15^MCM3T160A cells infected with PPV group,and the expression of VP2 of PPV was also significantly increased（P<0.05）.In this study,mouse polyclonal antibodies against VP2 or NS1 of PPV were successfully prepared respectively.The level of PVV replication increased rapidly,but the level of cellular DNA replication and the ability of MCM3 binding to histone H3 and MCM3binding to the DNA replication origins of cells both decreased remarkably with the PPV infection time prolonged in PK-15 cells.Furthermore,NS1 was the key viral proteins that suppressed the phosphorylation of 160th threonine of MCM3 after PPV infection.And the decrease of MCM3 phosphorylation promoted the VP2-MCM3 interaction,but suppressed the MCM3-histone H3 interaction.The MCM3^{T 160A},which we named PK-15^MCM3T160A cells,overexpression cell strains were constructed.When the phosphorylation of 160th threonine of MCM3 was mutated would considerably promote the replication of PPV.The results above elucidated the molecular mechanism of how VP2 hijacks MCM3 to promote viral replication,and provided a theoretical basis for further exploration of the pathogenic mechanism of PPV.