Study On The Mechanism Of BmNPV Iap1 Regulating The Cell Cycle Of Bombyx Mori

As intracellular parasites,viruses rely on cell adaptation and regulation to promote their replication and proliferation.Many viruses promote their own replication and proliferation,depending on regulating the cell cycle process of host,which is an important feature of their pathogenesis.Therefore,studying the relationship between virus and cell cycle has important biological significance for in-depth understanding of the interaction mechanism between host and virus.As one of the Lepidopteran model insects,Bombyx mori is an important insect due to the presence of silk.Bombyx mori nucleopolyhedrovirus(Bm NPV)is a DNA-like virus that specifically infects the silkworm.Moreover,the silkworm-virus infection model can be used as an experimental animal model to analyze the regulatory mechanism between virus infection and the host.Therefore,this paper aims to explore the relationship between Bm NPV and the cell cycle,in order to deepen the understanding of the pathogenesis of Bm NPV and the interaction network between Bm NPV and host.This study first analyzed the effect of Bm NPV infection on the cell cycle,and then determined that G2/M phase cells are conducive to the production for Bm NPV by measuring the effects of cells in different phase on the replication and proliferation of Bm NPV and the role of host DNA replication genes in Bm NPV DNA replication.Finally,throung immunoprecipitation and mass spectrometry,viral proteins that may interact with cell cycle factors were screened out,and then found that Bm NPV iap1 induced G2/M arrest by depleting Bm CDK1.Together,our research promotes understanding of the interaction mechanism between baculovirus and host,and provide a potential target for the prevention and treatment of Bm NPV.The main results and conclusions obtained in the paper are as follows:1.Effect of Bm NPV infection on the host cell cycleIn order to study the effect of Bm NPV infection on the cell cycle,we infected Bm NSWU1 cells with Bm NPV.Flow cytometry analysis confirmed that Bm NPV infection had no effect on the cell cycle before 9 h post-infection(h p.i.);the proportion of cells in G2/M phase increased by about 10% at 12 h p.i.,and it further increased by more than20% at 24 h p.i.and 48 h p.i.compared to uninfected cells,suggesting that the cell cycle was arrested in G2/M phase after 12 h p.i.M phase cells were observed by immunofluorescence.As a result,there are prophase,metaphase,anaphase and telophase cells in the mock group.While,the number of cells in anaphase and telophase significantly decreased at 12 h p.i.,and almost no cells in anaphase and telophase was observed at 24 h p.i.and 48 h p.i.,indicating that Bm NPV gradually blocked the cell cycle before metaphase after 12 h p.i.In order to explore the effect of Bm NPV infection on cell DNA replication,the Brd U-labeling method was used to observe and analyze the DNA replication during Bm NPV infection.The results showed that compared to uninfected cells,Bm NPV infection had no significant effect on cell DNA replication at 6 h p.i.,but significantly inhibited cell DNA replication at 48 h p.i.In addition,the results of cell proliferation activity test showed that cell proliferation activity gradually decreased after Bm NPV infection.Further,real-time fluorescent quantitative PCR and western blotting showed that during viral infection,the transcription and protein levels of Bm Cyclin B and Bm CDK1(the key genes converted from G2 to M)were significantly down-regulated;the localization of Bm CDK1 protein did not change significantly,while Bm Cyclin B protein entered the nucleus from the cytoplasm.The above results indicate that Bm NPV infection affects the host cell cycle,and blocks the cell cycle in G2/M phase.2.G2/M phase cells were favorable for replication and proliferation of Bm NPVIn order to prove the effect of cells in different phase on replication and proliferation of Bm NPV,cells numbers in G1 phase,S phase and G2/M phase were significantly increased by Aphidicolin,Hydroxyurea and Nocodazole,respectively.Then we detected the relative expression levels of the immediate early gene ie1 and viral genome copies.The results suggested that G2/M phase cells provides favorable environment and resources for replication and proliferation of Bm NPV;G1 and S phase cells significantly inhibited viral production.In order to exclude the effect of synchronization reagents on the Bm NPV,the cell cycle distribution,expression of viral gene ie1 and viral DNA replication were further analyzed in elution Aphidicolin and Nocodazole cells,respectively.The results showed that the uninfected cells in G1 phase gradually entered S phase with the elution of Aphidicolin,the proportion of S phase cells accounted for79.54% at Aphidicolin eluting 12 h p.i.,and then entered G2/M phase;with the elution of Nocodazole,the un-infected cells in the G2/M phase entered G1 phase and S phase,but the proportion of cells in G2/M phase still reached more than 50% within Nocodazole eluting 48 h.However,compared to un-infected cells,Bm NPV infection had no significant effect on cell cycle before 12 h p.i.in the Aphidicolin and Nocodazole eluted group,while the number of cells transformed from S phase to G2/M phase was about 26%less at 24 h p.i.and 48 h p.i.in the Aphidicolin eluted group,and Bm NPV infection arrested cell cycle in G2/M phase at 24 h p.i.and 48 h p.i.in the nocodazole eluted group.Furthermore,we detected the relative expression levels of viral gene ie1 and viral genome copies in the Aphidicolin and Nocodazole eluted cells.The results also showed that cells of G2/M phase were beneficial to replication and proliferation of Bm NPV,cells of G1 and S phase significantly inhibited viral production.3.Effect of host DNA replication genes on Bm NPV DNA replicationThe host completes DNA replication in S phase,while Bm NPV mainly replicates in G2/M phase cells.Therefore,this study further explores whether Bm NPV needs to utilize host cell-related replication proteins to complete its own replication.This study first identified the expression characteristics of host DNA replication genes during Bm NPV infection.It was found that Bm NPV infection down-regulated the relative expression levels of host DNA replication genes,such as MCM complex(Bm MCM4 and Bm MCM7),single strand DNA binding proteins(Bm RPA1 and Bm RPA2),proliferating cell nuclear antigen(Bm PCNA)and RFC complexes(Bm RFC3,Bm RFC4 and Bm RFC5).Bm MCM7 protein was mainly localized in the cytoplasm,and Bm RPA2,Bm RFC4,Bm RFC5 and Bm PCNA proteins co-localized with the virogenic stroma by Immunofluorescence.Bm NPV also encode some proteins needed for viral DNA replication,such as DNA polymerase(DNA poly)and single strand DNA binding proteins(lef3 and dbp).Therefore,real-time fluorescent quantitative PCR was used to further analyze the relationship between the host DNA replication genes and viral DNA replication genes.The results showed that overexpression of Bm RPA2,Bm RFC4,Bm RFC5 and Bm PCNA downregulated the relative expression levels of viral genes,DNA poly,lef3 and dbp,and the viral genome copies was significantly lower;RNAi of Bm RPA2,Bm RFC4,Bm RFC5 and Bm PCNA showed a significant higher viral genome copies;the overexpression of lef3 and dbp strikingly reduced the relative expression levels of Bm RPA1,Bm RPA2,Bm RFC3,Bm RFC4,Bm RFC5 and Bm PCNA.The above results indicate that DNA replication of Bm NPV dose not require the major proteins involved in host cell DNA replication.4.Bm NPV iap1 involve in regulating the cell cycleAccording to previous results,it was speculated that Bm NPV infection may induce G2/M phase arrest by regulating Bm Cyclin B and Bm CDK1 to promote self-proliferation and replication.In order to explore the molecular mechanism of G2/M arrest induced by Bm NPV,it was firstly investigated whether Bm Cyclin B and Bm CDK1 effected cell cycle and viral production.The results reflected that RNAi of Bm Cyclin B and Bm CDK1 significantly increased the numbers of G2/M phase cells and promoted the relative expression levels of viral genes,suggesting that Bm NPV infection arrested the cell cycle in G2/M by down-regulating the expression of Bm Cyclin B and Bm CDK1.In order to explore the molecular mechanism of Bm NPV infection affecting the changes of Bm Cyclin B and Bm CDK1,co-immunoprecipitation and mass spectrometry were used to screen viral proteins that may interact with Bm Cyclin B and Bm CDK1 during Bm NPV infection.Candidate viral proteins that may interact with Bm Cyclin B include Bm85,Bm67,Bm110,Bm NPV IAP1,Gta,Pe38 and He65,and candidate viral proteins that may interact with Bm CDK1 include Bm60,Bm10,ODV-E56,Bm NPV IAP1,Bm68 and Pe38.Among them,Pe38 and Bm NPV IAP1 were common candidate viral proteins.Immunofluorescence co-localization and co-immunoprecipitation confirmed that Bm NPV IAP1 interacted with Bm CDK1,and Bm Cyclin B interacted with Bm CDK1 during Bm NPV infection.To identify the regulation of Bm NPV iap1 on cell cycle,overexpression and knockout of Bm NPV iap1 were performed in Bm N-SWU1 cells.Overexpression of Bm NPV iap1 gene induced the host cell cycle arrest in G2/M phase,and had no effect on host DNA replication.Furthermore,the relationship of Bm NPV iap1 with Bm Cyclin B and Bm CDK1 was proved by real-time fluorescent quantitative PCR and immunoblot.The results showed that overexpression of Bm NPV iap1 gene resulted in the significant decrease of protein levels of Bm Cyclin B and Bm CDK1,albeit no effect on the transcriptional level of Bm Cyclin B and Bm CDK1.Knockout of Bm NPV iap1 significantly reduced the number of cells arrested in G2/M phase induced by Bm NPV.These results suggest that Bm NPV iap1 was related with G2/M phase arrest regulated by Bm NPV infection.Based on the above research results,we speculate the molecular mechanism of G2/M phase arrest induced by Bm NPV infection: after Bm NPV invades silkworm cells,Bm NPV iap1 promotes the reduction of Bm Cyclin B and Bm CDK1 protein content and interferes with the localization of Bm Cyclin B protein,and thereby Bm NPV gradually arrestes the cell cycle in G2/M phase.At the same time,Bm NPV inhibites the expression of host DNA replication genes via lef3 and dbp to promote its own DNA replication.