Study On Mechanisms Of Bombyx Mori Cytoplasmic Polyhedrosis Virus Transcription Based On The Cryo-EM 3D Reconstruction And Baculovirus Expression System

With the simplest structure(single layer capsid)and segmented double-strand RNA(ds RNA),Cytoplasmic Polyhedrosis Virus(CPV)is a representative virus of the Reoviridae family.Therefore,the study of CPV is of great significance.By using cryo-electron microscopy(cryo-EM)and 3D reconstruction,the structure of CPV has been revealed: CPV particle has 12 vertexes(turret),and icosahedron capsid consists with 60 asymmetric units,each asymmetric unit contains one turret protein(TP),2 large protrusion protein(LPP)and 2 capsid shell protein(CSP).Inside the capsid,10 TEC particularly binding with 10 turrets,and 10 segmented ds RNA binding with 10 TECs.Several active sites have been found on the TP protein,and these active sites can regulate the conformational changes of viral structural proteins.However,how these conformational changes affect the transcription and capping process of the virus remains a mystery.Using molecular biology and baculovirus expression system,we successfully expressed the TP and the m TP with 301 Tryptophan,302 Aspartic acid and 305 Tyrosine mutation.And using colorimetric method to test the substrate of TP and m TP,we identified the putative ATPase active site in the TP and demonstrated that 301 Tryptophan,302 Aspartic acid,and 305 Tyrosine play an important role in ATPase active site activity.In addition,we captured a near-atomic resolution in situ structure of Rd Rp by using cryo-EM 3D reconstruction,which reveals how the SAM,GTP,and ATP regulate the conformational changes of Rd Rp and prepares for the m RNA transcription.This study reveals a “signal pathway” within the CPV and explained the relationship between TEC conformational changes and viral transcription.These results also provide some ideas and references for the study of ds RNA viruses transcription mechanisms,drug development,and disease prevention.