Quantitative Proteomics Of Orf60-deleted BmNPV ODV And Acetylation Of Bm60

Bombyx mori nucleopolyhedrovirus(BmNPV),a member of alpha baculovirus Group I,is the most widely studied species that infect the silkworm,Bombyx mori.Bm60 is a late protein encoded by orf60 gene in the genome of BmNPV T3 strain.Previous studies have shown that:(1)When deleted the orf60 gene,it was observed that the production of budded virus(BV)was reduced by nearly an order of magnitude in BmN cells compared with that of wild type virus,and the virulence and shape of ODV particles were also impaired;(2)Protein acetylomics of cells infected with BmNPV T3 strain revealed that Bm60 was acetylated.This study mainly was focused on quantitative proteomics of orf60-deleted BmNPV ODV and the acetylation of Bm60.The main results were as follows:1.Quantitative proteomics analysis of orf60-deleted BmNPV ODV(1)Using the orf60-deleted bacmid(Bm60KO),the wild type bacmid(Bm60Rep)was constructed to express Bm60 fused with a Flag tag at the C-terminus.Western blot assays were performed with rabbit polyclonal antibody against P74/PIF0,PIF1,PIF2,PIF3,PIF5,ODV-E66 and VP39.The results showed that the expression of these viral proteins,except PIF1 and PIF3,were not impaired in infected cells upon deletion of the orf60 gene.(2)Quantitative proteomics of ODV showed that Bm60 is a component protein of ODV.When the orf60 gene was deleted,the amounts of several viral proteins located in the ODV envelope changed significantly.These proteins,the F-like protein,PIF3,Bm121 and CHITINASE that were related to viral pathogenicity decreased markedly.These results indicated that Bm60,as an ODV component protein,could impair the amount of envelope proteins on ODV,resulting in abnormal shape of ODV envelope and eventually disability of the virus to infect silkworm per os.2.The study of Bm60 acetylation(1)Recombinant bacmids and transient expression vectors to express Bm60 were constructed.It was determined by immunoprecipitation and cellular fractionation experiments that Bm60 was acetylated during viral infection and transient expression.(2)A series of deacetylated mimic(K/R)mutants expressing Bm60 were constructed.The results obtained by immunoprecipitation and Western blot found that an acetylated site was K236.(3)Acetylated mimic(K/Q)and deacetylated mimic(K/R)mutants expressing Bm60 protein were constructed.Then the virus growth curve was defined.The results showed that acetylation of K236 in Bm60 did not influence virus proliferation.