Global analysis of phosphorylation pattern of silkworm cells after BmNPV infection and the functional investigation of BmNPV 39K

摘要

Bombyx mori has become an important model organism for many fundamental studies.Bombyx mori nucleopolyhedrovirus(BmNPV)is a significant pathogen to Bombyx mori,yet also an efficient vector for recombinant protein production.A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive.Employing tandem mass tag(TMT)labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis,the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at24hpi with an MOI of 10was extensively examined.Totally,6480phosphorylation sites in2112protein groups were identified,among which4764sites in1717proteins were quantified.Among the quantified proteins,81up-regulated and 25down-regulated sites were identified with significant criteria(the quantitative ratio above1.3was considered as up-regulation and below0.77was considered as down-regulation)and with significant p-value(p＜0.05).Some proteins of BmNPV were also hyperphosphorylated during infection,such as P6.9,39K,LEF-6,Ac58-1ike protein,Ac82-1ike protein and BRO-D.The phosphorylated proteins were primary involved in several specific functions,out of which,we focused on the binding activity,protein synthesis,viral replication and apoptosis through kinase activity.
　　During infection,BmNPV39K has4phosphorylated sites which one of them has a great phosphorylation ratio(16.683).Interestingly,the homolog of39k,AcMNPV orf36also called pp31has been reported to be phosphorylated as well.On this basis,we then aimed to further investigate the function of phosphorylation on BmNPV39K in the viral replication and transcription.In order to investigate the biological function of phosphorylation in BmNPV39K,we constructed the mutant of the highest phosphorylated site of39K(136th amino acid)to be the positive and negative mutant.We then inserted these two mutants and wild type39K to the knocked out39K Bacmid by Lambda-red mediated repair gene technique.These three kinds of repaired Bacmid along with wild type and knocked out bacmid were then transfected to BmN cells and further investigated by qPCR analysis.
　　The result of qPCR showed that the BmNPV39K phosphorylation does not have significant effect on the viral replication.The viral transcriptional level investigation by qPCR showed that each type of viral gene;early gene(lef-3),late gene(vp39),and very late gene(p10)transcription were likely to be lessened in the cells that were transfeeted with39K positive mutant repaired Bacmid and vice versa in the cells transfected with39K negative mutant repaired Bacmid.The positive mutation that was done to mimic the phosphorylation could reduce the viral transcription while the negative mutation to induce de-phosphorylation has ability to elevate it suggesting that phosphorylation on BmNPV39K tends to alleviate the viral transcription in each phase.
　　In conclusion,BmNPV39K is an early gene in which the phosphorylation of this protein will not be an essential mechanism for DNA viral replication.Interestingly,the phosphorylation of BmNPV39K apparently plays an important role in the viral transcription.However,from this result we could yield an assumption that BmNPV39K phosphorylation which diminishes the viral transcription is a mechanism required by the virus to prolong the life of cells in order to harness the cellular material for the production of their viral progeny.Moreover,there is also a possibility that BmNPV39K phosphorylation is an important pathway regulated by the cellular enzyme to protect themselves from the onset of virus.This study will lay the groundwork for further investigation in the function of BmNPV39K phosphorylation on the viral genome replication and transcription.

目录

声明

Abstract

Abbreviation

Contents

Chapter 1：Background of phosphorylation

1．Phosphorylation in living things

2．Phosphorylation in Bombyx mori and BmNPV

3．Phosphorylation in BmNPV 39K

Chapter 2：Research Program

1．Purpose and significance

2．Research method

3．Workflow design

Chapter 3：Global analysis of phosphoproteome pattern of silkworm cells after BmNPV infection

2．2 Sample preparation

2．3 Quantitative proteomic analysis by LC-MS／MS

2．4 Bioinformatics analysis

3．Results

3．1 Quantification of phosphoproteome

3．2 Bioinformatics analysis

4．Discussion

4．1 Phosphorylation may have a pivotal function on the regulation of binding activity

4．2 Phosphorylation may represent an obligatory regulation in protein synthesis

4．3 Phosphorylation may be responsible for viral replication during infection

4．4 Phosphorylation may regulate anti-apoptotie cells through the kinases activity after BmNPV infection

4．5 The phosphorylation of BmNPV protein

5．Conclusions

Chapter 4：The functional investigation of BmNPV 39K phosphorylation

1．Bioinformatics analysis of 39K

1．1 Tools

1．2 Methods

1．3 Results and discussion

2．Generation of mutant strain

2．1 Materials and reagents

2．2 Methods

2．3 Results

3．The investigation of point mutation effect at S136 phosphosite ofBmNPV 39K

3．1 Material and reagents

3．2 Methods

3．3 Results

4．Discussion

5．Conclusion

References

Acknowledgment