Research On The Function Of BmNPV Orf90 Gene In Bombyx Mori Nucleopolyhedrovirus

BmNPV(Bombyx mori Nucleopolyhedrovirus)is the typical representative of Nucleopolyhedrovirus.In recent years,the exploration of BmNPV gene function has never stopped,but there are still a lot of unknown function genes.Bm NPV orf90(Bm90)is a highly conserved gene in the genome of BmNPV,but its function remains unknown.In this study,we presented the effect of Bm90 on the replication,transcription and assembly of BmNPV in the whole virus infection cycle.The Bm90 knockout virus was constructed by ?Red recombination system and the Bm90 repair virus was constructed by Bac-to-Bac system.Meanwhile,egfp and polh genes were inserted to different bacmid to constructed three bacmids(WtBacmid-polh-egfp,Bm90ko-polh-egfp-Bacmid and Bm90re-polh-egfp-Bacmid).The three bacmids DNA were respectively used to transfect BmN cells.The TCID50 value of Bm90ko-polh-egfp-Bacmid remained at zero,indicating that the knockout of Bm90 resulted in a defect in production of infectious budded virus(BV).Transmission electron microscopy(TEM)analysis was used to further analyze whether the deletion of Bm90 interferes with the assembly.BmN cells were transfected with three types viral and examined by TEM at 48 h post transfection.Nuclei of cells transfected with either WtBacmid-polh-egfp or Bm90re-polh-egfpBacmid DNA exhibited the typical baculovirus infection symptoms such as an extensive number of nucleocapsids and reorganized,electron dense virogenic stroma.However,no nucleocapsids or other typical baculovirus infection symptoms appeared in the cells transfected with Bm90ko-polh-egfp-Bacmid DNA,indicating that Bm90 is required for the assembly of nucleocapsids.qPCR analysis found that the DNA replication levels generated by WtBacmid-polh-egfp and Bm90re-polh-egfp-Bacmid transfected cells showed a similarly steady increase from 12 h to 96 h post transfection.However,there was no evident increase of DNA replication levels from 48 h to 96 h post transfection in Bm90ko-polh-egfp-Bacmid transfected cells.Therefore,qPCR analysis suggests that Bm90 is not required for the onset of viral DNA replication in BmN cells.Meanwhile,qRT-PCR analysis reveals that Bm90 deletion exerts a strong down-regulatory effect on transcription levels of early gene lef-3,late gene vp39,and very late gene p10(P<0.05),indicates that Bm90 may regulated other virus gene expression in the baculoviral life cycle.In addition,we constructed the Bm90 protein expression vector pET28a-Bm90 and the fusion protein was purified.A polyclonal antibody prepared from the New Zealand white rabbits infused with a purified fusion protein.Bm90 protein was detected in BmN cells after 72 h post transfection by Western blot,indicates that Bm90 gene might initiate the expression during the late phase of viral life.