Global Analysis Of Phosphorylation Pattern Of Silkworm Cells After BmNPV Infection And The Functional Investigation Of BmNPV 39K

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 at 24 hpi with an MOI of 10 was extensively examined.Totally,6480 phosphorylation sites in 2112 protein groups were identified,among which 4764sites in 1717 proteins were quantified.Among the quantified proteins,81 up-regulated and 25down-regulated sites were identified with significant criteria?the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was 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-like protein,Ac82-like 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,BmNPV 39K has 4 phosphorylated sites which one of them has a great phosphorylation ratio?16.683?.Interestingly,the homolog of 39k,AcMNPV orf 36 also called pp31 has been reported to be phosphorylated as well.On this basis,we then aimed to further investigate the function of phosphorylation on BmNPV 39K in the viral replication and transcription.In order to investigate the biological function of phosphorylation in BmNPV 39K,we constructed the mutant of the highest phosphorylated site of 39K(136^th amino acid)to be the positive and negative mutant.We then inserted these two mutants and wild type 39K to the knocked out 39K 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 BmNPV 39K 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 transfected with 39K positive mutant repaired Bacmid and vice versa in the cells transfected with 39K 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 Bm NPV 39K tends to alleviate the viral transcription in each phase.In conclusion,BmNPV 39K 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 BmNPV 39K apparently plays an important role in the viral transcription.However,from this result we could yield an assumption that BmNPV 39K 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 BmNPV 39K 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 BmNPV 39K phosphorylation on the viral genome replication and transcription.