Studies On The Symmetry-mismatch Structures Of DsRNA Virus By Cryo-EM Single Particle

Viruses widely exist in every corner of the biosphere,and their hosts include vertebrates,invertebrates,plants,algae,fungi,yeasts and even prokaryotes,which are closely related to human life activities and social life.Since 1898,Dutch bacteriologist Beilink took the lead in identifying the hospital of tobacco Mosaic disease as "a filtering pathogenic factor",human's research on viruses has a long history of nearly one and a half centuries,and human's understanding of viruses has also risen from the vague concept to the precise description of molecular biology level.In recent years,more and more virus microstructure is nearly complete analytical atomic resolution,thanks to the continuous improvement and perfection of structural biology research tools,including hon Xian chicken stand is not frozen electron Microscopy Technology(then electron Microscopy Technology,then the EM).Its high degree of generality,fidelity and accuracy has been recognized by the majority of experts and scholars in the industry.It has won the honor of "Method of the Year" in 2015 by the journal Nature Methods.In2020,together with "Virus",it was once again awarded the "Top 10 Scientific Discover of the Year" by the journal Nature.The life cycle of viruses has long been the focus of research.Up to now,with the development and update of X-ray Crystallography and Single Particle Analysis of cryoEM,a large number of viral capsid structures have been analyzed at the level of near atomic resolution,which greatly accelerates the process of human understanding,using and modifying viruses.The method of analyzing the capsid structure of icosahedral virus by single particle technique of cryo-electron microscopy has become more and more mature.However,the virus is not a simple icosahedral structure,and there is a problem of local deformation under the irradiation damage of electron beam.The purpose of this work is to complete the structural analysis of the symmetrical mismatch proteins of double-stranded RNA viruses with icosahedral symmetrical structure by means of three-dimensional reconstruction of symmetric mismatch by freezing electron microscopy,and then propose the molecular biological mechanism of reasonable life cycle of double-stranded RNA viruses.Grass Carp Reovirus belonging to the genus Aquareovirus is selected as the research object of this project.This virus is a double-stranded RNA virus with double capsid,with strong virulence and wide influence range.It is a very representative research model of double-stranded RNA viruses.This topic of the whole process of research on natural degradation(Native Degraded,ND)condition,Trypsin(Trypsin Treated Particle,TTP)of viral capsid structure parsing,respectively.Then,based on the high-resolution capsid structure and the conformational changes occurring in different states,the molecular biological mechanisms related to virus attachment,invasion and assembly were proposed.On the basis of high resolution capsid structure,the icosahedral symmetric mismatch protein and genome structure were analyzed by symmetric mismatch algorithm respectively,and the high resolution symmetric mismatch protein structure was obtained.Based on these structures,the molecular biological mechanism of viral transcription,replication and assembly was proposed.Based on the above methods,this study obtained the following results:(1)The icosahedral symmetry structure of grass carp reovirus under natural degradation state was analyzed,and its 3.2A near-atomic resolution structure was obtained.Icosahedral symmetry mismatch algorithm was used to obtain the resolution structure of the whole virus particle 3.4A,and the conformational difference of the capsid protein VP3 A under the 5-fold Axis was found.At the same time,A high resolution structure of RNA polymerase VP2 and its synergistic VP4 complex with Pseudo D3-symmetry was obtained,with A resolution of 3.6A.The atomic model was constructed based on the effective density map.Then the molecular mechanisms of viral transcription and assembly were proposed based on the above structures.(2)The capsid protein of grass carp reovirus under trypsin treatment was analyzed to obtain the capsid structure with A resolution of 4.2A.On this basis,the local threedimensional reconstruction method was used to analyze the pentamer Turret Protein at the five axis of symmetry and the surrounding VP5-VP7 trimer Protein complex,and the resolution structure of 3.84 A was obtained.With natural degradation of grass carp reovirus 5 times near the axis of symmetry of conformational differences and conformation of main electrical conduction VP3 A loci,put forward A virus that USES icosahedron symmetric mismatch algorithm to obtain the whole virus particles 3.4-a resolution structure,and found the capsid protein VP3 A conformational differences under five axis of symmetry.(3)For the missing location of RdRp complex in the virus,an algorithm based on local asymmetric orientation search and global asymmetric orientation search was designed,and a preliminary structural model was developed.(4)Combined with the above structural information,the molecular biology of viral quasi-transcriptional state is described,and the molecular biological mechanism of transcription process of ds RNA viruses is improved.The intracellular assembly mechanism of ds RNA viruses is proposed.The mechanism of virus host cell attachment is proposed.The possibility of mechanism of virus from dormant state to active state is proposed.